mRNA vaccines for epstein-BARR virus (EBV) and diseases caused by EBV infection

Abstract

Described herein are compositions and methods for treating or preventing EBV infection in a subject, and treating or preventing a disease or disorder caused by EBV infection in a subject.

Description

[0001] Attorney Docket No. 11538-008W01

[0002] MRNA VACCINES FOR EPSTEIN-BARR VIRUS (EBV) AND DISEASES CAUSED BY

[0003] EBV INFECTION

[0004] CROSS-REFERENCE TO RELATED APPLICATION

[0005] This application claims priority to, and the benefit of U.S. Provisional Application 63 / 694,02.2, filed on September 12, 2024, the contents of which is hereby incorporated in its entirety.

[0006] REFERENCE TO SEQUENCE LISTING

[0007] The Sequence Listing submitted September 12, 2025, as a text filed named “11538- 008W01JST26” created September 12, 2025, and having a file size of 152,563 bytes is hereby incorporated by reference pursuant to 3'7 C.F.R. § 1.52(e)(5).

[0008] BACKGROUND

[0009] The Epstein Barr virus (EBV) is a member of the g-herpesvirus family, viruses that are characterized by the establishment of lifelong infection, most frequently in a latent form, and Subsequent activation leading to a lytic state occurs in response to poorly understood signals and can be manifested by the production of virus, leading to downstream disease sequelae. It is estimated that there is near-universal presence of EBV in humans, with at least 95% of human adults infected worldwide. EBV is the etiologic agent in infectious mononucleosis, chronic active EBV infection, multiple sclerosis, and EBV- associated hemophagocytic lymphohistiocytosis, and is the presumed etiologic agent in diseases of immune dysregulation, including periodontitis and rheumatoid arthritis. EBV is also the first identified oncogenic vires in humans and is identified as a group 1 carcinogen by the World Health Organization (Wong Y et al.. Estimating the Global Burden of Epstein-Ban' Virus-Related Cancers. J Cancer Res Clin Oncol (2022) 148(1):31— 46.). EB V is linked to at least 1.5% of all cases of human malignancies worldwide (Klein G. Tumor Associations of EBV -Historical Perspectives. Curr Top Microbiol Immunol (2015) 390(Pt l):17-22.). The association of EBV infection -specific cancers includes nasopharyngeal carcinoma (NPC), gastric cancer, Hodgkin’s lymphoma, and Burkitt’s lymphoma.

[0010] Similar to the other Herpesviridae in this class, EBV virus production from latently infected host tissue unfolds in a multistep process that relies on the expression of multiple internal and surface-expressed viral glycoproteins. Attorney Docket No. 11538-008W01

[0011] Lytic gene products are produced in three consecutive stages: immediate-early, early, and late. Immediate-early lytic gene products act as transactivators, enhancing the expression of later lytic genes. Reactivation of the virus from latency depends on expression of the viral immediate- early gene, BamHI Z fragment leftward open reading frame 1 (BZLF1). The BZLF1 promoter normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore, histone deacetylase inhibitor, or anti-Ig. (Regulation of Epstein-Barr virus reactivation from latency Takayuki Murata, Microbiol Immunol 2014; 58: 307-317).

[0012] In latency, EBV proteins are expressed in one of three latency programs: Latency I, II, or III. Each latency program leads to the production of a set of viral proteins and viral RNAs. (Calderwood MA, et al. (May 2007). Epstein-Barr virus and virus human protein interaction maps, PNAS. 104 (18): 7606-11. Hutzinger R, et al. (August 2009). "Expression and processing of a small nucleolar RNA from the Epstein-Barr virus genome". PLOS Pathogens. 5 (8): 61000547). The latency II program produces EBNA1, LMP1 and LMP2A. In epithelial cells, LMP1 induces the expression of anti-apopt otic proteins and cell surface antigens. In EBV infected junctional epithelial cells, LMP1, and BZLF1 expression are readily detected (Journal of Oral Biology, 2024, VOL. 16, 2301199.), making them putative vaccine targets for T cell immunity, to eliminate infected cells possibly before they become laden with virions. LMP1 is also a potent activator of IL-8, an inflammatory mediator of human chronic periodontitis via the accumulation and degranulation of neutrophils, which causes the subsequent destruction of periodontium (in vivo 33: 1793-1800, (2019)).

[0013] EBV virions exhibit dual tropism, infecting mainly epithelial cells and B cells. The EBV virion utilizes complex and seemingly redundant cellular entry mechanisms, with specific glycoproteins seemingly restricted to assisting entry into distinct cell types. EBV gp350 plays an important role in B-cell infection, as EBV binds to and infects naive human B lymphocytes via CD21, the CR2 complement receptor. This binding rapidly activates the NF-KB transcription factor (J Exp Med. 1997; 186(5): 731-737), which, in turn, binds to and mediates transcriptional activation of Wp, the initial viral latent gene promoter. Thus, EBV binding to its cellular receptor on resting B cells triggers an NF-kB-dependent intracellular signaling pathway, which is an early step toward. Glycoproteins gp350, gp42, gH / gL heterodimers, and gB, appear to be important to the infection of B cells. Glycoprotein BMRF2, gH / gL, and gB are involved in virus binding, entry, and fusion in epithelial cells. EBV gH / gL and gB participate in the infection of both B cells and epithelial cells and together comprise the core fusion machinery of EBV. EBV gB is the viral fusion protein, fusion being a common theme in viral pathophysiology, and Attorney Docket No. 11538-008W01 represents the core component of the fusion machinery wi th a striking amount of structural homology across the Herpesviridae. Amongst the surface glycoproteins, gB is the most highly conserved glycoprotein in sequence and structure. EBV gB also plays a role in the maturation of EBV virions, and the egress of virus from the cell. Interestingly, EBV lacking the gB genes cannot be produced. Thus, gB is a key target for vaccines as antibodies to the gB protein can disrupt viral maturation, egress, and entry into host cells (reviewed in Hong J, et al, Glycoprotein B Antibodies Completely Neutralize EBV Infection of B Cells. Front. Immunol. 2022, 13:920467).

[0014] The presence of EBV-DNA in dental tissues is commonly associated with chronic periodontitis. Several investigators have validated the role of EBV in the etiopathogenesis of chronic periodontitis (CP) (Vincent -Bugnas S, et al. (2013) EBV Infection Is Common in Gingival Epithelial Cells of the Periodontium and Worsens during Chronic Periodontitis. PLoS ONE 8(12): e80336.). CP is a common inflammatory disease of the gums and recognized as the major cause of gum disease, inflammation and tooth loss. CP results in the destruction of the alveolar bone and the deepening of the gingival sulcus crevice, leading to tire formation of pathologically deep periodontal pockets (PP). The depth of the PP correlates with periodontal disease progression. In fact, the amount of EBV DNA detected in PPs correlates with disease severity. The formation and presence of ectopic lymphoid follicles in the gums are indicative of the role that ongoing EBV virion production plays in driving the immunopathology, and, not surprisingly, are seen in other EBV-related immune dysregulation-driven diseases, including multiple sclerosis and rheumatoid arthritis. Thus, EBV actively replicates in periodontal epithelial cells, causing both host pathology and leading to transmission via shedding.

[0015] There is a need for compositions and methods to prevent EBV infection or treat EBV caused diseases.

[0016] The compositions and methods disclosed herein address these and other needs.

[0017] SUMMARY

[0018] Described herein are compositions including: a nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 , EB V gBPF or a variant or fragment thereof including SEQ. ID. NO: 2, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8, EBV gL-Linker-gH or a variant or fragment thereof including SEQ.

[0019] ID. NO: 7, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 , EBV prefusion gB or a variant or Attorney Docket No. 11538-OO8WQ1 fragment thereof including SEQ. ID. NO: 29, SEQ, ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36. SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof.

[0020] Described herein are compositions including: a nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof, wherein the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP-2A, EBV LMP-2B, EBV EBN A- 1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLE-1, or a combination thereof.

[0021] Described herein are compositions including: a first nucleic acid encoding for Epstein Barr virus (EBV) glycoprotein BMRF2 or a variant or fragment thereof; and a second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L (gL), EBV glycoprotein 1, (gL) EBV glycoprotein gp350, or a combination thereof.

[0022] Described herein are compositions including: a first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 , EB V gBPF or a variant or fragment thereof including SEQ. ID. NO: 2, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8, EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 , EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ, ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof; and a second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L (gL), EBV glycoprotein I, (gL) EBV glycoprotein gp350, or a combination thereof.

[0023] Described herein are also compositions including: a first nucleic acid encoding for an Epstein Barr virus (EBV) envelope protein or a variant or fragment thereof; and a second nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof.

[0024] Described herein are nucleic acids including SEQ. ID. NO: 4 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. Attorney Docket No. 11538-008W01

[0025] Described herein are ceils including a nucleic acid including SEQ. ID. NO: 4, a protein described herein, or the composition described herein.

[0026] Described herein are methods of treating or preventing EBV infection in a subject, the methods including: administering to the subject a therapeutically effective amount of the compositions described herein or nucleic acid including SEQ. ID. NO: 4 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity.

[0027] Described herein are also methods of preventing a disease or disorder caused by EBV infection in a subject, the method including: administering to the subject a therapeutically effective amount of the composition described herein or the nucleic acid including SEQ. ID. NO: 4 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity.

[0028] Also described herein are methods of treating a disease or disorder caused by EBV infection in a subject, the method including: administering to the subject a therapeutically effective amount of the composition described herein or the nucleic acid including SEQ. ID. NO: 4 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity.

[0029] DESCRIPTION OF DRAWINGS

[0030] FIGs. 1 A-1F show EBV gp350 mRNA LNP vaccine candidates formulated with ARV-T1 induced markedly high titers of neutralizing antibodies and strong T cell immune response in BALB / c mice. (1 A) and (IB) show in vitro gp350 expression after transfection of 293T cells with gp350FL mRNA / LNP and gp470 mRNA / LNP. Flow cytometry analysis of EBV gp350 expression after transfection of 293T cells with gp350FL LNP, gp470T LNP, or transfection of 293T cells with gp350FL mRNA and gp470T mRNA using Lipofectamine MessengerMAX transfection reagent. (1A) Percentage of gp350 positive cells. (IB) Median fluorescence of gp350 positive cells. (1C) show immunization with gp350FL LNP and gp470 LNP induced high titers of gp350-specfic antibodies (binding antibodies). BALB / c mice were immunized twice on days 0 and 21, and sera were obtained on days 21 and 35 for the analysis of gp350- specific antibodies with ELISA. * p<0.05, ** p<0.01 , *** p<0.005, **** p<0.001 compared to mice immunized with lower dose the same LNP. # p<0.05, ## p<0.01, ### p<0.005, #### p<0.001 compared to mice immunized with the same dose but different LNP (gp350FL versus gp470). (I D) and (IE) show' immunization with gp350FL 1..NP and gp470 LNP induced high titers of EBV neutralizing antibodies. ID. Day 35 immune sera were analyzed for EBV neutralizing Attorney Docket No. 11538-008W01 antibodies with AKATA cells and EBV expressing GFP. * p<0.05 compared to mice immunized with lower dose the same LNP. IE. EBV neutralization antibodies elicited by gp350 nanoparticles published by Dr. Jeffery Cohen’s group at NIH. **** p<0.001. (IF) show EBV gp350FL LNP and gp470 LNP elicited strong T cell response with IFN-y production. BALB / c mice spleen cells were obtained 35 days post immunization for the analysis IFN-y production via ELISpot after stimulation with Ipg / ml recombinant gp350 overnight. * p<0.05 compared to mice immunized with lower dose the same LNP.

[0031] FIGs. 2A-2E show EBV gH / gL mRN A LNP vaccine candidates formulated with ARV-T1 induced potent antibody and T cell immune responses in BALB / c mice. (2A) and (2B) show in vitro EBV gH / gL expression after transfection of 293T cells with gH / gL mRNAs or gH / gL mRNA-LNPs. Flow cytometry analysis of EBV gH / gL expression after transfection of 293T cells with gH+gL mRNA LNP, gL-(G4S)3-gH (SEQ. ID. NO: 7) mRNA LNP or gL-T2A-H (SEQ. ID. NO: 8) mRNA LNP, or transfection of 293T cells with gH+gL mRNA or gL-(G4S)3- gH mRNA using Lipofectamine MessengerMAX mRNA transfection reagent. (2A) percentage of gH / gL positive cells. B(2) median fluorescence of gH / gL. positi ve cells. (2C) show's immunization wdth gH+gL mRNA LNP and gL-(G4S)3-gH mRNA LNP induced high titers of gH / gL-spectic antibodies (binding antibodies). BALB / c mice were immunized twice on days 0 and 21, and sera were obtained on days 21 and 35 for the analysis of gH / gL-specific antibodies with ELISA. * p<0.05, ** p<0.01, ♦** p<0.005, **♦* p<0.001 compared to mice immunized with lower dose the same LNP. ## pcO.Ol, compared to mice immunized with the same dose but different LNP (gH+gL LNP versus gL-(G4S)3-gH). (2D) shows immunization with gH+gL mRNA LNP and gL-(G4S)3-gH mRNA LNP induced high titers of EBV neutralizing antibodies. Day 35 immune sera were obtained for the analysis of EBV neutralizing antibodies with AKATA cells and an EBV expressing GFP. * p<0.05, ** pcO.Ol, *** p<0.005 compared to mice immunized with lower dose the same LNP. (2E) shows EBV gH+gL mRNA LNP and gL-(G4S)3- gH mRNA LNP elicited strong T cell response with IFN-y production. BALB / c mouse spleen cells were obtained 35 days post immunization for the analysis IFN-y production via ELISpot after stimulation with Ipg / ml recombinant gH / gL protein overnight. * p<0.05, ** pcO.Ol , *** p<0.005 compared to mice immunized with lower dose the same LNP. # p<0.05, compared to mice immunized with the same dose but different LNP (gH+gL mRNA LNP versus gL-(G4S)3- gH mRNA LNP).

[0032] FIGs. 3A-3D show EBV gB mRNA LNP vaccine candidates formulated with ARV-T1 induced high titers gB-specific antibodies and potent T cell immune response in BALB / c mice. Attorney Docket No. 11538-008W01

[0033] (3 A) shows in vitro EBV gB expression after transfection of 293T cells with EBV gBWT (SEQ. ID. NO: 1), gBLN (SEQ. ID. NO: 3) or gBPF (SEQ. ID. NO: 2) mRNA-LNP. Lane 1, 293 T cells as negative control. Lane 2, 293T cells transfected with EBV gBWT mRNA-LNP. Lane 3, 293T cells transfected with EBV gBLN mRNA-LNP. Lane 4, 293T cells transfected with EBV gBPF mRNA-LNP. (3B) shows immunization with EBV gBWT, gBLN or gBPF mRNA-LNP induced high titers of gB-specfic antibodies (binding antibodies). BALB / c mice were immunized twice on days 0 and 21, and sera were obtained on days 21 and 35 for the analysis of gB- specific antibodies with ELISA. * p<0.05, ** p<0.01, *** p<0.005, **♦* pcO.OOl compared to mice immunized with lower dose the same LNP. # p<0.05, ## p<0.01 , ### p<0.005, #### p<0.001 compared to mice immunized with the same dose but different LNP (gBWT versus gBLN versus gBPF). (3C) shows immunization with EBV gBWT, gBLN or gBPF mRNA-LNP elicited moderate titers of EBV neutralizing antibodies. BALB / c mice were immunized twice on days 0 and 21, and sera were obtained on day 35 for the analysis of EBV neutralizing antibodies with AKATA cells and EBV expressing GFP. (3D) shows EBV gBWT, gBLN and gBPF mRNA- LNPs all elicited strong T cell response with IFN-y production. BALB / c mice were immunized twice on days 0 and 21, and spleen were obtained on day 35 for the analysis IFN-y production via ELISpot after stimulation with Ipg / ml recombinant gB protein overnight.

[0034] FIGs. 4A-4D show prefusion EBV gB mRNA LNP vaccine candidates formulated with ARV-T1 induced high titers of EBV gB binding antibodies and EBV neutralizing antibodies as well as potent T cell immune response in BALB / c mice. (4A) shows in vitro EBV gB expression after transfection of 293T cells with EBV EBTMH, EBPRH, EBMPR and EBMPR-V mRNA- LNPs. Lane 1, 293 T cells as negative control. Lane 2, 293T cells transfected with EBV EBTMH mRNA-LNP. Lane 3, 293T cells transfected with EBPRH mRNA-LNP. Lane 4, 293T cells transfected with EBMPR mRNA-LNP. Lane 5, 293T cells transfected with EBMPR-V mRNA- LNP. (4B) shows immunization with EBV EBTMH, EBPRH, EBMPR and EBMPR-V mRNA- LNPs induced high titers of gB-specfic antibodies (binding antibodies). BALB / c mice were immunized twice on days 0 and 21, and sera were obtained on days 21 and 35 for the analysis of gB-specific antibodies with ELISA. (4C) shows immunization with EBV EBPRH, EBMPR and EBMPR-V mRNA-LNPs elicited high titers of EBV neutralizing antibodies. BALB / c mice were immunized twice on days 0 and 21, and sera were obtained on day 35 for the analysis of EBV neutralizing antibodies with AKATA cells and EBV expressing GFP. (4D) shows EBV EBTMH, EBPRH, EBMPR and EBMPR-V mRNA-LNPs all elicited strong T cell response with IFN-y production. BALB / c mice were immunized tv, dee on days 0 and 21, and spleen were obtained on Attorney Docket No. 11538-008W01 day 35 for the analysis IFN-y production via ELISpot after stimulation with 1 ug / ml recombinant gB protein overnight.

[0035] FIGs. 5A-5C show co-transfection of EBV gB mRNA LNP with gH / gL mRNA LNP as well as co-formulation of gB mRNA and gH / gL mRNA in a single LNP resulted in the expression of EBV gB / gH / gL complex. (5 A) shows in vitro EB V gB / gH / gL complex expression after co-transfection of 293T cells with EBV gB mRNA-LNP [EBNOP (SEQ. ID. NO: 3), EBTMH and EBPRM] with EBV gL-(Gly4Ser)3-gH mRNA-LNP. Lane 1, 293 T cells as negative control. Lane 2, 293T cells co-transfected with EBTMH mRNA-LNP and gL- (Gly4Ser)3-gH mRNA-LNP. Lane 3, 293T cells co-transfected with EBV EBPRM mRNA-LNP and gL -(Gly4Ser)3-gH mRNA-LNP. Lane 4, 293T cells co-transfected with EBNOP mRNA- LNP and gL -(Gly4Ser)3-gH mRNA-LNP. (5B) shows in vitro EBV gB / gH / gL complex expression after co-transfection of 293T cells with EBV gB mRNA-LNP (EBPRH, EBMPR and EBMPR-V) with EBV gL-T2A-gH mRNA-LNP. Lane 1, 293 T cells as negative control. Lane 2, 293T cells co-transfected with EBPRH mRNA-LNP and EBV gL-T2A-gH mRNA-LNP. Lane 3, 293T cells co-transfected with EBV EBMPR mRNA-LNP and EBV gL-T2A-gH mRN A-LNP. Lane 4, 293T cells co-transfected with EBMPR-V mRNA-LNP and EBV gL-T2A-gH mRNA- LNP. (5C) shows in vitro EBV gB / gH / gL complex expression after transfection of 293T cells with a single LNP encapsulating EBV gB mRNA-LNP and gH / gL mRNA. Lane 1, 293 T cells as negative control. Lane 2, 293T cells transfected with a single LNP encapsulating EBNOP mRN A and gL-(Gly4Ser)3-gH mRNA. Lane 3, 293T cells transfected with a single LNP encapsulating EBTMH mRNA and gL-(Gly4Ser)3-gH mRNA. Lane 4, 293T cells transfected with a single LNP encapsulating EBTMM mRNA and gL-(Gly4Ser)3-gH mRNA. Lane 5, 293T cells transfected with a single LNP encapsulating EBPRH mRNA and gL-T2A-gH mRNA mRNA. Lane 6, 2933" cells transfected with a single LNP encapsulating EBPRM mRNA and gL-T2A-gH mRNA mRNA. Lane 7, 293T cells transfected with a single LNP encapsulating EBMPR mRNA and gL- T2 A-gH mRNA mRNA.

[0036] FIGs. 6A-6B show EBV BZLF1 mRNA LNP vaccine candidates formulated with ARV- T'l induced potent T cell immune responses in C57BL / 6 mice. (6A) shows in vitro EBV BZLF1 expression after transfection of 293T cells with EBV BZLFlwt or BZLFlopt mRNA-LNP. Lane 1, 293 T cells as negative control. Lane 2, 293T cells transfected with EBV BZLFlwt mRNA- LNP. Lane 3, 293T cells transfected with EBV BZLFlopt mRNA-LNP. (6B) shows EBV BZLFlwt and BZLFlopt mRNA-LNPs both elicited strong T cell response with IFN-y production. C57BL / 6 mice were immunized twice on days 0 and 21, and spleen cells were Attorney Docket No. 11538-008W01 obtained on day 35 for the analysis IFN-y production via ELISpot after stimulation with 2.5pg / ml BZLF1 peptide mix overnight.

[0037] FIGs. 7A-7B show EBV EBNA1 mRNA LNP vaccine candidates formulated with ARV- T1 induced potent T cell immune responses in C57BL / 6 mice. (7 A) shows in vitro EBV EBNA1 expression after transfection of 293T cells with EBNAlwt or EBNAldel mRNA-LNP. Lane 1, 293 T cells as negative control. Lane 2, 293T cells transfected with EBV EBNAlwt mRNA- LNP. Lane 3, 293T cells transfected with EBNAldel mRNA-LNP. (7B) shows EB V EBNAldel mRNA-LNPs elicited strong T cell response with IFN-y production. C57BL / 6 mice were immunized twice on days 0 and 21, and spleen were obtained on day 35 for the analysis IFN-y production via ELISpot after stimulation with 2.5pg / ml EBNA1 peptide mix overnight.

[0038] FIG. 8 shows EBV LMP1 mRNA LNP vaccine candidates formulated with ARV-T1 showed high level protein expression in vitro. Lane 1, 293 T cells as negative control. Lane 2, 293T cells transfected with EBV LMPldel mRNA-LNP. Lane 3, 293T cells transfected with LMPlwt mRNA-LNP.

[0039] FIG. 9 shows EBV LMP2 mRNA LNP vaccine candidates formulated with ARV-T1 showed high level protein expression in vitro. Lane 1, 293 T cells as negative control. Lane 2,

[0040] 293T cells transfected with EBV LMP2wt mRNA-LNP. Lane 3, 293T cells transfected with

[0041] LMP2mut mRNA-LNP.

[0042] Attorney Docket No. 11538-008W01

[0043] Like reference symbols in the various drawings indicate like elements.

[0044] DETAILED DESCRIPTION

[0045] Epstein-Barr virus (EBV) is a gamma human herpesvirus, it is the primary cause of infectious mononucleosis, and a recent epidemiology study showed evidence that EBV is also the cause of multiple sclerosis. EBV is also the first human tumor virus discovered and is strongly implicated in the etiology of multiple lymphoid and epithelial cancers. Additional studies demonstrated that EBV plays key roles in the pathogenesis of other diseases and disorders, including chronic periodontitis, rheumatoid arthritis, and systemic lupus erythematosus.

[0046] Described herein are nucleic acid (e.g., mRNA) vaccines that elicit immunity targeting EBV proteins for the prevention and treatment of EBV infections. The vaccines described herein are designed to encode specific antigens of EBV to elicit a robust, functional, and potentially synergistic immune response targeting key EBV antigens and to elicit specific immune responses to key virus proteins that manifest at different and key stages of the progression from latent to lytic cycles, binding and viral fusion thereby disrupting the production of virus and subsequent inflammation.

[0047] The nucleic acid vaccine compositions are described that can be administered both prophylactically to prevent EBV infection as well therapeutically to treat EBV infection as well as diseases caused by EBV, such as multiple sclerosis, EBV associated cancers, chronic immune dysregulation, chronic periodontitis, among others.

[0048] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0049] Definitions

[0050] To facilitate understanding of the disclosure set forth herein, a number of terms sire defined below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0051] General Definitions

[0052] As used in this specification and the following claims, the terms “comprise” (as well as Attorney Docket No. 11538-008W01 forms, derivatives, or variations thereof, such as “comprising” and “comprises”) and “include” (as well as forms, derivatives, or variations thereof, such as “including” and “includes”) are inclusive (i.e., open-ended) and do not exclude additional elements or steps. For example, the terms "comprise" and / or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Other than where noted, all numbers expressing quantities of ingredients, reaction conditions, geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

[0053] Accordingly, these terms are intended to not only cover the recited element(s) or step(s), but may also include other elements or steps not expressly recited. Furthermore, as used herein, the use of the terms “a”, “an”, and “the” when used in conjunction with an element may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Therefore, an element preceded by “a” or “an” does not, without more constraints, preclude the existence of additional identical elements.

[0054] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. By “about” is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range i s expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. A range may be constmed to include the start and the end of the range. For example, a range of 10% to 20% (i.e., range of 10%-20%) can includes 10% and also includes 20%, and includes percentages in between 10% and 20%, unless explicitly stated otherwise herein.

[0055] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may Attorney Docket No. 11538-008W01 include an excipient" is meant to include cases in which the formulation inchides an excipient as well as cases in which the formulation does not include an excipient.

[0056] It is understood that when combinations, subsets, groups, etc. of elements are disclosed (e.g., combinations of components in a composition, or combinations of steps in a method), that while specific reference of each of the various individual and collective combinations and permutations of these elements may not be explicitly disclosed, each is specifically contemplated and described herein.

[0057] “Administration" to a subject includes any route of introducing or deli vering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, transcutaneous, transdermal, intra-joint, intra-arteriole, intradermal, intraventricular, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra- articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. "Concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. "Systemic administration" refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject's body (e.g. greater than 50% of the body ), for example through entrance into the circulatory or lymph systems. By contrast, "local administration" refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another.

[0058] As used here, the terms “beneficial agent” and “active agent” are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned Attorney Docket No. 11538-008W01 herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the terms “beneficial agent” or “active agent” are used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0059] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0060] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, tire reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0061] “Inactivate”, “inactivating” and “inactivation” means to decrease or eliminate an activity, response, condition, disease, or other biological parameter due to a chemical (covalent bond formation) between the ligand and a its biological target.

[0062] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.

[0063] As used herein, the terms “treating” or “treatment” of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms “treating” and “treatment” can also refer to reduction in severity and / or frequency of symptoms, elimination of symptoms and / or underlying cause, prevention of the occurrence of symptoms and / or their underlying cause, and improvement or remediation of damage. Attorney Docket No. 11538-008W01

[0064] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms “prevent” or “suppress” can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms. As used herein, the term “preventing” a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and / or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event.

[0065] By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount”. However, an appropriate “effective’ amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.

[0066] An “effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0067] As used herein, a “therapeutically effective amount” of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a “prophylactically effective amount” of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of a Attorney Docket No. 11538-008W01 given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term ‘'therapeutically effective amount” can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and / or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.

[0068] As used herein, the term “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0069] "Pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "earner" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0070] As used herein, “pharmaceutically acceptable salt” is a derivative of the disclosed compound in which the parent compound is modified by making inorganic and organic, nontoxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water Attorney Docket No. 11538-008W01 or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.

[0071] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic sal ts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, H00C-(CH2)n- COOH where n is 0-4, and the like, or using a different acid that produces the same counterion. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0072] Also, as used herein, the term “pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0073] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0074] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a veterinary patient. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a human.

[0075] Genetic Definitions

[0076] The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides or ribonucleotides. Attorney Docket No. 11538-008W01

[0077] The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

[0078] The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribon ucleotides .

[0079] The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers of from about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of doublestranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry, Third Ed., (1988), incorporated herein by reference for all purposes.

[0080] The term “polynucleotide” refers to a single or double stranded polymer composed of nucleotide monomers. The polynucleotide sequence may be modified, for example, to enhance efficacy and / or to reduce immune responsivity, by using, for example, base modifications or endcapping. In other embodiments, an unmodified polynucleotide sequence is used. For example, the polynucleotide can be an RNA sequence or a DNA sequence. In some embodiments, the mRNA can include an optimized codon. By codon optimizing, the formation of secondary structures can be reduced and translational efficiency improved. In certain embodiments, the codon optimization includes GC enrichment of the coding region. In certain embodiments, the codon optimization includes codon quality enrichment of the coding region. In certain aspects, the mRNA can include one or more regions or parts, which act or function as an untranslated region (UTRs) of a gene. UTRs are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. The 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The use of human-derived UTRs may facilitate the expression of the polypeptide in cells. In some embodiments, the polynucleotide comprises at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically Attorney Docket No. 11538-008W01 modified phosphodiester linkage, or a combination thereof. In some embodiments, the polynucleotide sequence as used comprise modified nucleosides such as 5-methylcystonsine or psudouridine.

[0081] As used herein “modified” refers to a changed state or structure of a molecule of the invention. Molecules may be modified in many ways including chemically, structurally, and functionally. In one embodiment, the polynucleotides of the present invention are “chemically modified” by the introduction of non-natural nucleosides and / or nucleotides, e.g., as it relates to the natural ribonucleotides A, U, G, and C. Modifications of the nucleosides and / or nucleotides as used in the present invention may be naturally occurring (i.e. comprise a nucleotide and / or nucleoside other titan the natural ribonucleotides A, U, G, and C) or may be artificial. Non- canonical nucleotides such as the cap structures are not considered “modified” although they differ from the chemical structure of A, G, C, and U ribonucleotides. As used herein, a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. When the polynucleotides of the present invention are chemically and / or structurally modified, the polynucleotides may be referred to as “modified nucleotides”.

[0082] In some embodiments, the nucleic acids disclosed herein can include at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof.

[0083] In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase.

[0084] In one embodiment, the chemically modified nucleobase is selected from 5- formylcytidine (5fC), 5-methylcytidine (5meC), 5-methoxyeytidine (5moC), 5-hydroxycytidine (5hoC), 5-hydroxymethylcytidine (5hmC), 5 -formyluridine (5fU), 5-methyluridine (5-meU), 5- methoxyuridine (5moU), 5-carboxymethylesteruridine (5camU), pseudouridine (T), Nl- methylpseudouridine (melT), N6-methyladenosine (me6A), or thienoguanosine (thG).

[0085] In some embodiments, the chemically modified nucleobase is 5-methoxyuridine (5moU). In some embodiments, the chemically modified nucleobase is pseudouridine (T). In some embodiments, the chemically modified nucleobase is Nl-methylpseudouridine ( me IT).

[0086] The structures of these modified nucleobases are shown below: Attorney Docket No. 11538-008W01

[0087] In one embodiment, the at least one chemically modified nucleotide is a chemically modified ribose.

[0088] In one embodiment, the chemically modified ribose is selected from 2 ’ -O-methyl (2 ' - O-Me), 2' -Fluoro (2' -F), 2' -deoxy-2' -fluoro-beta-D-arabino-nucleic acid (2 ' F-ANA), 4 ' -S, 4' -SFANA, 2' -azido, UNA, 2 ' -O-methoxy-ethyl (2 ' -O-ME), 2' -O- Allyl, 2' -O- Ethylamine, 2 ' -O-Cyanoethyl, Locked nucleic acid (LAN), Methylene-cLAN, N-MeO-amino BNA, or N-MeO-aminooxy BNA. In one embodiment, the chemically modified ribose is 2 '' -O- methyl (2 ' -O-Me). In one embodiment, the chemically modified ribose is 2 ' -Fluoro (2 ' -F).

[0089] The structures of these modified riboses tire shown below:

[0090] Attorney Docket No. 11538-008W01

[0091] In one embodiment, the at least one chemically modified nucleotide is a chemically modified phosphodiester linkage.

[0092] In one embodiment, the chemically modified phosphodiester linkage is selected from phosphorothioate (PS), boranophosphate, phosphodithioate (PS2), 3 ' ,5 ' -amide, N3 ' - phosphoramidate (NP), Phosphodiester (PO), or 2 ’ ,5 ’ -phosphodiester (2 ' ,5 ' -PO). In one embodiment, the chemically modified phosphodiester linkage is phosphorothioate.

[0093] The structures of these modified phosphodiester linkages are shown below:

[0094] Attorney Docket No. 11538-008W01

[0095] In some embodiments, the mRNA can include a heterologous 5’ untranslated region (5'UTR). In some embodiments, the mRNA can include a heterologous 3’ untranslated region (3’UTR).

[0096] The term “polypeptide” refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds. A polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids which are not members of the standard twenty amino acids may also be used. The standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Tip, W), tyrosine (Tyr, Y), and valine (Vai, V). The terms ’’polypeptide sequence" or “amino acid sequence” are an alphabetical representation of a polypeptide molecule.

[0097] Conservative substitutions of amino acids in proteins and polypeptides are known in the art. For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations such as, for example, Gly, Ala; Vai, He, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the polypeptides provided herein. Attorney Docket No. 11538-008W01

[0098] Substantial changes in protein function or immunological identity are made by selecting substitutions that are less conservative, i.e., selecting residues that differ more significantly in their effect on maintaining (a) die structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and / or glycosylation.

[0099] A “variant” refers to a molecule substantially similar in structure. Thus, in one embodiment, a variant refers to a protein whose amino acid sequence is similar to a reference amino acid sequence, but does not have 100% identity with the respective reference sequence. The variant protein has an altered sequence in which one or more of the amino acids in the reference sequence is deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the alterations, the variant protein has an amino acid sequence which is at least 60%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the reference sequence. For example, variant sequences which are at least 95% identical have no more than 5 alterations, i.e. any combination of deletions, insertions or substitutions, per 100 amino acids of the reference sequence.

[0100] The term “complementary” refers to the topological compatibility or matching together of interacting surfaces of a probe molecule and its target. Thus, the target and its probe can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.

[0101] The term “hybridization” refers to a process of establishing a non-covalent, sequencespecific interaction between two or more complementary strands of nucleic acids into a single hybrid, which in the case of two strands is referred to as a duplex.

[0102] The terms “identical” or percent “identi ty,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably Attorney Docket No. 11538-008W01

[0103] 74%, 75%, 76%, / 7%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences sire then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0104] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0105] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when Attorney Docket No. 11538-008W01 aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues: always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulati ve score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or tire end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=~4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M-5, N=-4, and a comparison of both strands.

[0106] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0107] The term "nucleobase" refers to the part of a nucleotide that bears the Watson / Crick basepairing functionality. The most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.

[0108] Compositions

[0109] Described herein are compositions including: a nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least Attorney Docket No. 11538-008W01

[0110] 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV BMRF2 or a variant or fragment thereof including SEQ. ID.

[0111] NO: 11 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0112] In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EB V envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL- T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least Attorney Docket No. 11538-008W01

[0113] 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0114] In some embodiments, the composition can further include a nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof. In some embodiments, the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP- 2A, EBV LMP-2B, EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1, or a combination thereof.

[0115] In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic Attorney Docket No. 11538-008W01 protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

[0116] In some embodiments, the nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof includes SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof includes SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof includes SEQ. ID. NO: 16 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof includes SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof includes SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 41, SEQ. ID. NO: 42, or a variant having at least 90% (e.g. 90%, 91 %. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof includes SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof includes SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof includes SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the Attorney Docket No. 11538-008W01 nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof includes SEQ. ID. NO: 27, SEQ. ID. NO: 28, or a variant having at least 90% (e.g. 90%', 91%, 92%, 93%, 94%, 95%, 96%, 979o, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0117] In some embodiments, the composition can further include a nucleic acid encoding for EBV gB or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gH or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gL or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof.

[0118] In some embodiments, the second nucleic acid encoding for EBV gB or a variant or fragment thereof includes SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ, ID. NO: 31, SEQ. ID. NO: 32, SEQ, ID. NO: 33, SEQ. ID. NO: 34.SEQ. ID. NO: 35. SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV gH or a variant or fragment thereof includes SEQ. ID. NO: 5. In some embodiments, the nucleic acid encoding for EBV gL or a variant or fragment thereof includes SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV gp350 or a variant or fragment thereof includes SEQ. ID. NO: 9, SEQ. ID. NO: 10, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0119] Described herein are compositions including: a nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof, wherein tire EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1 , EBV LMP-2A, EBV LMP-2B, EBV EBNA- 1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1, or a combination thereof.

[0120] In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a Attorney Docket No. 11538-008W01 nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EB V immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EB V BZLF-1 or a variant or fragment thereof.

[0121] In some embodiments, the nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof includes SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof includes SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EB V LMP-2B or a variant or fragment thereof includes SEQ. ID. NO: 16 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof includes SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-2 or a variant or Attorney Docket No. 11538-008W01 fragment thereof includes SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 41, SEQ. ID. NO: 42, or a variant having at least 90% (e.g. 90%, 91 %. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof includes SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof includes SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof includes SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV BZLF-1 or a vari ant or fragment thereof includes SEQ. ID. NO: 27, SEQ. ID. NO: 28, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%', 97%, 98%, 99%', 99.8%, or 99.9%) identity, or a combination thereof.

[0122] Described herein are compositions including: a first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 , EB V gBPF or a variant or fragment thereof including SEQ. ID. NO: 2, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8, EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 , EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ, ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof; and a second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L (gL), EBV glycoprotein I, (gL) EBV glycoprotein gp350, or a combination thereof.

[0123] In some embodiments, the first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least Attorney Docket No. 11538-008W01

[0124] 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the first nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30. SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0125] In some embodiments, the second nucleic acid can include a nucleic acid encoding for EBV gB or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gH or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gL or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof.

[0126] In some embodiments, the second nucleic acid encoding for EBV gB or a variant or fragment thereof includes SEQ. ID. NO: 1. SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ, ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, Attorney Docket No. 11538-008W01

[0127] 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV gH or a variant or fragment thereof includes SEQ. ID. NO: 5. In some embodiments, the nucleic acid encoding for EBV gL or a variant or fragment thereof includes SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV gp350 or a variant or fragment thereof includes SEQ. ID. NO: 9, SEQ. ID. NO: 10, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0128] In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least 90%' (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-Linker- gl-I or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EB V prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ. ID. NO: 33. SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0129] In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBPF or a variant or fragment thereof including SEQ. Attorney Docket No. 11538-008W01

[0130] ID. NO: 2 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%. 99.8%, or 99.9%) identity. In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL-Linker-gH or a variant or fragment thereof inchiding SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof

[0131] In some embodiments, the composition can further include a nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof. In some embodiments, the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP- 2A, EBV LMP-2B, EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EB V BZLF-1, or a combination thereof.

[0132] In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment Attorney Docket No. 11538-008W01 thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV L.MP-2B or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof. In some embodiments, the nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof includes a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

[0133] In some embodiments, the nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof includes SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof includes SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof includes SEQ. ID. NO: 16 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof includes SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination Attorney Docket No. 11538-008W01 thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof includes SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ, ID. NO: 41, SEQ. ID. NO: 42, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof includes SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof includes SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof includes SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV BZLF-1 or a vari ant or fragment thereof includes SEQ. ID. NO: 27, SEQ. ID. NO: 28, or a variant having at least 90%' (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0134] Described herein are compositions including: a first nucleic acid encoding for Epstein Barr virus (EBV) glycoprotein BMRF2 or a variant or fragment thereof; and a second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L (gL), EBV glycoprotein L (gL) EBV glycoprotein gp350, or a combination thereof.

[0135] In some embodiments, the first nucleic acid includes SEQ. ID. NO: 11.

[0136] In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gB or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gH or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gL or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof.

[0137] In some embodiments, the second nucleic acid encoding for EBV gB or a variant or fragment thereof includes SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ, ID. NO: 36, SEQ. ID. NO: 37, SEQ, ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV gH or a variant or fragment thereof Attorney Docket No. 11538-008W01 includes SEQ. ID. NO: 5. In some embodiments, the nucleic acid encoding for EBV gL or a variant or fragment thereof includes SEQ. ID. NO: 6, SEQ. ID. NO: 7. SEQ. ID. NO: 8, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV gp350 or a variant or fragment thereof includes SEQ. ID. NO: 9, SEQ. ID. NO: 10, or a variant having at least 90% (e.g. 90%, 91%, 92%', 93%, 94%, 95%', 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0138] In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91%, 92%, 939o, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-Linker- gH or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ, ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0139] In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least 90% (e.g. 90%, 91%, 92%', 93%, 94%, 95%', 96%, 97%, 98%', 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV Attorney Docket No. 11538-OO8WQ1 envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL- T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least 90% (e.g. 90%, 91%, 92%', 93%, 94%, 95%', 96%, 97%, 98%', 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof inchides a nucleic acid encoding for EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90%' (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) Identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof inchides a nucleic acid encoding for EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31. SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0140] In some embodiments, the composition further includes a third nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof, wherein the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP-2A, EBV LMP-2B. EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1, or a combination thereof.

[0141] In some embodiments, the third nucleic acid includes a variant of EBV latent protein and / or EBV immediate early lytic protein including a deletion / mutation to one or more amino acid residues to eliminate oncogenic function. In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof. In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof. In some embodiments, the third nucleic acid includes a nucleic acid Attorney Docket No. 11538-008W01 encoding for EBV LMP-2B or a variant or fragment thereof. In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof.

[0142] In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof. In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof. In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof. In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof. In some embodiments, the third nucleic acid includes a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

[0143] In some embodiments, the third nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof includes SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the third nucleic acid encoding for EB V LMP-2A or a variant or fragment thereof includes SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the third nucleic acid encoding for EB V L.MP-2B or a variant or fragment thereof includes SEQ. ID. NO: 16 or a variant having at least 90% (e.g. 90%. 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity,. In some embodiments, the third nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof includes SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the third nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof includes SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 41 , SEQ. ID. NO: 42, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the third nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof includes SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof. In some embodiments, the third nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof includes SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%', 94%, 95%, 96%', 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the third nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof includes SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a variant having at least 90% (e.g. 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the third nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof Attorney Docket No. 11538-008W01 includes SEQ. ID. NO: 27, SEQ, ID. NO: 28, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0144] Described herein are also compositions including: a first nucleic acid encoding for an Epstein Barr virus (EBV) envelope protein or a variant or fragment thereof; and a second nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof.

[0145] In some embodiments, the EBV envelope protein or a variant or fragment thereof is selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L (gL), EBV glycoprotein H (gH), EBV glycoprotein gp350. EBV glycoprotein BMRF2, or a combination thereof. In some embodiments, the first nucleic acid includes a nucleic acid encoding for EBV glycoprotein BMRF2 or a variant or fragment thereof. In some embodiments, the first nucleic acid includes a nucleic acid encoding for EBV gB or a variant or fragment thereof. In some embodiments, the first nucleic acid includes a nucleic acid encoding for EBV gH or a variant or fragment thereof. In some embodiments, the first nucleic acid includes a nucleic acid encoding for EBV gL or a variant or fragment thereof. In some embodiments, the first nucleic acid includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof.

[0146] In some embodiments, the nucleic acid encoding for EBV glycoprotein BMRF2 includes SEQ. ID. NO: 11. In some embodiments, the nucleic acid encoding for EBV gB or a variant or fragment thereof includes SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38. SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV gH or a variant or fragment thereof includes SEQ. ID. NO: 5. In some embodiments, the nucleic acid encoding for EBV gL or a variant or fragment thereof includes SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the nucleic acid encoding for EBV gp350 or a variant or fragment thereof includes SEQ. ID. NO: 9, SEQ. ID. NO: 10, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0147] In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gB WT or a variant or Attorney Docket No. 11538-008W01 fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gL-Linker- gH or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34.SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0148] In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL- T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: Attorney Docket No. 11538-008W01

[0149] 9 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: 11 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof includes a nucleic acid encoding for EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38. SEQ. ID. NO: 39, SEQ. ID. NO: 40 or a variant having at least 90% (e.g. 90%, 91%, 92%, 939o, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP-2.A, EBV LMP-2B, EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1, or a combination thereof. In some embodiments, the second nucleic acid includes a variant of EBV latent protein and / or EBV immediate early lytic protein including a deletion / mutation to one or more amino acid residues to eliminate oncogenic function.

[0150] In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof. In some embodiments, the second nucleic acid includes a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

[0151] In some embodiments, the second nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof includes SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, 99.8%, or 99.9%) identity, or a Attorney Docket No. 11538-008W01 combination thereof. In some embodiments, the second nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof includes SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof includes SEQ. ID. NO: 16 or a variant having at least 90%;(e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity. In some embodiments, the second nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof includes SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%. 94%, 95%, 96%. 97%, 98%, 99%. 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof includes SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 41, SEQ. ID. NO: 42, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof includes SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof includes SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof includes SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof. In some embodiments, the second nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof includes SEQ. ID. NO: 27, SEQ. ID. NO: 28, or a variant having at least 90% (e.g. 90%, 91%, 92%', 93%, 94%, 95%>, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity, or a combination thereof.

[0152] Also, described herein are nucleic acids including: SEQ. ID. NO: 4or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity,. Described herein are also cells including a nucleic acid including SEQ. ID. NO: 4, a protein described herein, or the composition described herein. In some embodiments, the cell includes a dendritic cell, B cell, or a macrophage. Attorney Docket No. 11538-008W01

[0153] In some embodiments, the nucleic acids described herein can include plasmid DNA, minicircle DNA, microRNA, mRNA, self-amplifying RNA, circle RNA, DNA launched selfamplifying RNA, or viral vector.

[0154] The compositions described herein can further include a suitable delivery vehicle. Suitable nucleic acid delivery vehicles are known in the art and can include, but are not limited to lipid-based (e.g., a liposome formulation, lipoplexes, or lipid nanoparticles (LNP)), viralbased, or physical methods such as injection, microinjection, electroporation, ultrasound, gene gun, hydrodynamic applications, or any combination thereof. In some embodiments, the composition includes a nanoparticle, a lipid nanoparticle dispersion, a liposomal formulation, a lipid emulsion, vaccine, vector, or any combination thereof.

[0155] Nanoparticles

[0156] In some embodiments, the nanoparticle can be a lipid nanoparticle. In some embodiments, the nanoparticles can be a lipid-polycation complex, referred to as a cationic lipid nanoparticle. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and / or polyarginine. In some embodiments, the lipid nanoparticle can include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0157] A lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Nature Biotech. 201028: 172-176), the lipid nanoparticle can further include 57% cationic lipid, 7% dipalmitoylphosphatidylcholine, 34% cholesterol, and 1.5% PEG-c-DMA. As another example, changing the composition of the cationic lipid can more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200).

[0158] In some embodiments, lipid nanoparticle formulations may comprise 35 to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid and / or 55% to 65% cationic lipid. In some embodiments, the ratio of lipid to nucleic acid (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30: 1 and / or at least 30:1.

[0159] Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid. Attorney Docket No. 11538-008W01

[0160] In some embodiments, the lipid nanoparticle includes 20% to 80% of ionizable lipid, cationic lipid, or any combination thereof: 0 % to 5 % pegylated lipids; 0 % to 40 % helper lipids; and 0 % to 80 % sterol.

[0161] In some embodiments, a lipid nanoparticle formulation comprises at least one ionizable lipid, cationic lipid, or any combination thereof; a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic lipid:5-25% neutral lipid:25-55% sterol; 0.5- 15% PEG-lipid.

[0162] In some embodiments, a lipid nanoparticle formulation includes 25% to 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)- non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)beptadecanedioate (L319), e.g., 35 to 65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.

[0163] In some embodiments, lipid nanoparticle formulations include 25-75% of a cationic lipid, 0.5-15% of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG or PEG-modified lipid on a molar basis.

[0164] In some embodiments, lipid nanoparticle formulations include 35-65% of a cationic lipid, 3-12% of the neutral lipid, 15-45% of the sterol, and 0.5-10%' of the PEG or PEG-modified lipid on a molar basis.

[0165] In some embodiments, lipid nanoparticle formulations include 45-65% of a cationic lipid,

[0166] 5-10% of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG or PEG-modified lipid on a molar basis.

[0167] In some embodiments, lipid nanoparticle formulations include 60% of a cationic lipid, 7.5% of the neutral lipid, 31% of the sterol, and 1 .5% of the PEG or PEG-modified lipid on a molar basis.

[0168] In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid, 10% of the neutral lipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modified lipid on a molar basis.

[0169] In some embodiments, lipid nanoparticle formulations include 50% of a cationic lipid, 10% of the neutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modified lipid, and 0.5% of the targeting lipid on a molar basis.

[0170] In some embodiments, lipid nanoparticle formulations include 40% of a cationic lipid, 15% of the neutral lipid, 40% of the sterol, and 5% of the PEG or PEG-modified lipid on a molar basis. Attorney Docket No. 11538-008W01

[0171] In some embodiments, lipid nanoparticle formulations include 57.2% of a cationic lipid, 7.1%' of the neutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modified lipid on a molar basis.

[0172] In some embodiments, lipid nanoparticle formulations include 57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. (J. Controlled Release, 107, 276-287 (2005), the contents of which are herein incorporated by reference in their entirety), 7.5% of the neutral lipid, 31.5% of the sterol, and 3.5% of the PEG or PEG-modified lipid on a molar basis.

[0173] In some embodiments, lipid nanoparticle formulations including a lipid mixture in molar ratios of 20-70% cationic lipid:5-45% neutral lipid:20-55% cholesterol: 0.5-15% PEG-modified lipid. In some embodiments, lipid nanoparticle formulations including a lipid mixture in a molar ratio of 20-60% cationic lipid:5-25% neutral lipid: 2.5-55% cholesterol: 0.5-15% PEG-modified lipid.

[0174] In some embodiments, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG- DPG), 57.2 / 7.1 / 34.3 / 1.4 (mol % cationic lipid / neutral lipid, e.g., DPPC / Chol / PEG- modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG- modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0.5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DSG), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 40 / 10 / 40 / 10 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG- cDMA) or 52 / 13 / 30 / 5 (mol % cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

[0175] Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2.012), Angew. Chem. Int. Ed., 51: 8529-8533; and Mater et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which tire incorporated herein by reference in their entirety).

[0176] In some embodiments, lipid nanoparticle formulations may comprise a cationic lipid, a PEG lipid and a structural lipid and optionally comprise a non-cationic lipid. As a non-limiting example, a lipid nanopartide may comprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid. As another non-limiting example, the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and Attorney Docket No. 11538-008W01

[0177] 38.5% structural lipid. As yet another non-limiting example, a lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.

[0178] In some embodiments, the lipid nanoparticle formulations described herein may be 4 component lipid nanoparticles. The lipid nanoparticle may comprise a cationic lipid, a noncationic lipid, a PEG lipid and a structural lipid. As a non-limiting example, the lipid nanoparticle may comprise 40-60%' of cationic lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structural lipid. As another non-limiting example, the lipid nanoparticle may comprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yet another non-limiting example, the lipid nanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5% structural lipid.

[0179] In some embodiments, the lipid nanoparticle formulations described herein may comprise a cationic lipid, a non-cationic lipid, a PEG lipid and a structural lipid. As a non-limiting example, the lipid nanoparticle comprises 50% of the cationic lipid DLin-KC2-DMA, 10% of the non-cationic lipid DSPC, 1.5%' of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprises 50% of the cationic lipid DLin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid cholesterol. As a non-limiting example, the lipid nanoparticle comprises 50% of the cationic lipid DLin-MC3-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of the structural lipid cholesterol. As yet another non-limiting example, the lipid nanoparricle comprises 55% of the cationic lipid L319, 10% of the non-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of the structural lipid cholesterol.

[0180] In some embodiments, the nanoparticles may be such as those described in U.S, Patent No. 10,933,127, the contents of which is herein incorporated by reference in its entirety. In some embodiments, the nanoparticles may be such as those described in U.S. Patent No. 10,933,127, for example, nanoparticles including a compound according to Formula (I), (la), (II), (Ila), (lib), (lie), (lid), or (He) described from column 101 to column 187.

[0181] In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm. The lipid nanoparticles (LNP) described herein may be made in a sterile environment. In some embodiments, the LNP formulation may be formulated in a nanoparticle such as a nucleic acid-lipid particle. As a non-limiting example, the lipid particle may comprise one or more active agents or therapeutic agents: one or more cationic lipids comprising from 50 mol % Attorney Docket No. 11538-008W01 to 85 mol % of the total lipid present in the particle; one or more non-cationic lipids comprising from 13 mol % to 49.5 mol % of the total lipid present in the particle; and one or more conjugated lipids that inhibit aggregation of particles comprising from 0.5 mol % to 2 mol % of the total lipid present in the particle.

[0182] The nanoparticle formulations may comprise a phosphate conjugate. The phosphate conjugate may increase in vivo circulation times and / or increase the targeted delivery of the nanoparticle. As a non-limiting example, the phosphate conjugates may include a compound of any one of the formulas described in International Application No. WO2013033438, the contents of which are herein incorporated by reference in its entirety.

[0183] The nanoparticle formulation may comprise a polymer conjugate. The polymer conjugate may be a water soluble conjugate. The polymer conjugate may have a structure as described in U.S. Patent Application No. 20130059360, the contents of which are herein incorporated by reference in its entirety. In some embodiments, polymer conjugates with the polynucleotides of the present disclosure may be made using the methods and / or segmented polymeric reagents described in U.S. Patent Application No. 20130072709, the contents of which are herein incorporated by reference in its entirety. In some embodiments, the polymer conjugate may have pendant side groups comprising ring moieties such as, but not limited to, the polymer conjugates described in U.S. Patent Publication No. US20130196948, the contents which are herein incorporated by reference in its entirety.

[0184] The nanoparticle formulations may comprise a conjugate to enhance the delivery of nanoparticles of the present disclosure in a subject.

[0185] In some embodiments, compositions comprising a nucleic acid described herein and a conjugate may have a degradable linkage. Non-limiting examples of conjugates include an aromatic moiety comprising an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer. As a non-limiting example, pharmaceutical compositions comprising a conjugate with a degradable linkage and methods for delivering such pharmaceutical compositions are described in U.S. Patent Publication No. US20130184443, the contents of which are herein incorporated by reference in their entirety.

[0186] Neutral Lipids

[0187] In some embodiments, a lipid nanoparticle formulation includes 0.5% to 15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include, without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments, the formulation includes 5% to 50%' on a molar basis of the sterol (e.g., 15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. A non-limiting example of a Attorney Docket No. 11538-008W01 sterol is cholesterol. In some embodiments, a lipid nanoparticle formulation includes 0.5% to 20% on a molar basis of the PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1 ,500 Da, around 1 ,000 Da, or around 500 Da. Non- limiting examples of PEG-modified lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which are herein incorporated by reference in their entirety).

[0188] Zwitterionic Lipids

[0189] In some embodiments, the composition may be encapsulated in, linked to and / or associated with zwitterionic lipids. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No. US20130216607, the contents of which are herein incorporated by reference in their entirety. In some aspects, the zwitterionic lipids may be used in the liposomes and lipid nanoparticles described herein.

[0190] Cationic Lipids

[0191] Suitable cationic lipids may include, but are not limited to, 2-amino-3-[(9Z,12Z)- octadec a-9 , 12-dien- 1 -yloxy] ■ 2- { [(9Z,2Z) -octadeca-9 , 12 ■ d ien- 1 -yloxy] methyl } propan ■ 1 -ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-l -yloxy]-2-{[(9Z)-octadec-9- en-l-yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)- octadeca-9,12-dien-l-yloxy]-2-[(octyloxy)methyl]propan-l-ol (Compound 3 in U S20130150625); and 2-(dimethyIamino)-3- [(9Z,12Z)-octadeca-9,l 2-dien- 1 -yloxyj-2- ( [(9Z,12Z)-octadeca-9,12-dien- l-yloxy]methyl }propan-l-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof. As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)~— N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z) — N,N-dimemylhexacosa- 17,20-dien-9-amine, ( 1Z, 19Z) — N5N-dimethylpentacosa- 16, 19-dien-8-amine, (13ZJ 6Z) — N,N-dimethyldocosa-l 3,16-dien-5-amine, (12Z,15Z) — N,N- dimethylhenicosa- 12, 15-dien-4-amine, ( 14Z, 17Z) — N,N-dimethyltricosa- 14,17 -di en-6-amine, ( 15Z, 18Z)--N,N -dimethyltetracosa- 15,18 -dien-7 -amine, ( 18Z,21Z)--N ,N-dimethyIheptacosa- 18,21-dien-10-amine, (15Z,18Z) — N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z) — N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z) — N,N-dimeihyloctacosa-19,22-dien-9- amine, (18Z.21 Z) — N,N-dimethylheptacosa-18,21-dien-8 amine, (17Z,20Z) — N,N- dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)---N,N-dimethylpentacosa-16,19-dien-6- Attorney Docket No. 11538-008W01 amine, (22Z..25Z) — N,N-dimethylhentriaconta-22,25-dien-10-amine, (21 Z..2.4Z) — N,N- dimethyltriaconta-21.24-dien-9-amine, (18Z) — N,N-dimetylheptacos-18-en-10-amine, (17Z) — N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-- N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z) — N-etbyl-N-methylnonacosa-20,23-dien-10- amine, 1 -[(11Z, 14Z)-1 -nonylicosa-ll,14-dien-l-yi]pyrrolidine, (20Z) — N,N-dimethylheptacos- 20-en-10-amine. (15Z) — N,N-dimethyI eptacos-15-en-10-amine, (14Z) — N,N-dimethylnonacos- 14-en-10-amine, (17Z)— N,N-dimethylnonacos-17-en-10-amine, (24Z)— -N,N- dimethyltritriacont-24-en-10-amine, (20Z) — N,N-dimethylnonacos-20-en-10-amine, (22Z) — N,N-dimethylhentriacont-22-en-10-amine, (16Z) — N,N-dimethylpentacos-16-en-8-amine, (12Z, 15Z) — N,N-dimethyl-2-nonylhenicosa- 12, 15-dien- 1 -amine, (13Z, 16Z) — N,N-dimethyl-3- nonyldocosa- 13,16-dien- 1 amine, N ,N-dimethyl- 1 - [(1 S ,2R)-2-octylcyclopropyl]eptadecan - 8- amine, l-[(lS,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-l- [(1 S,2R)-2-octylcyclopropyi]nonadecan-10- amine, N,N-dimethyl-21 -[(lS,2R)-2- octylcyclopropyl]henicosan-10-amine, N,N-dimethyl-l-[(lS,2S)-2-[[(lR,2R)-2- pentylcycIopropyl]niethyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-l-[(lS,2R)-2- octylcyclopropy 1 ]hexadecan -8 -amine, N,N-dimethyl- [( 1 R ,2S )-2-undecyicyclopropyl] tetradecan - 5-amine, N,N-dimethyl-3-{7-[(lS,2R)-2-octylcyclopropyl]heptyl}dodecan-l -amine, 1-[(1R,2S)~ 2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, l-[(lS,2R)-2-decylcyclopropyI]-N,N- dimethylpentadecan-6-amine, N ,N-dimethyl- 1 -R1 S ,2R)-2-octylcyclopropyllpentadecan-8- amine, R — N,N-dimethyl- 1 -[(9Z, 12Z)-octadeca-9, 12-dien - 1 -yloxy]-3-(octyioxy (propaneamine, S — N,N-dimetbyl-l-[(9Z,12Z(-octadeca-9,12-dien-l-yloxy]-3-(octyloxy(propan-2-amine, l-{2-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-l-[(octyloxy)methyl]ethyl)pyrrolidine, (2S)--N,N- dimethyl-1-[(9Z,l2Z)-octadeca-9,l2-dien-l-yloxyj-3-[(5Z)-oct-5-en-l-yloxy]propan-2-amine, 1- ( 2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy] - 1 - [(octyloxy (methyl] ethyl } aze ti dine, (2 S)- 1 - (hexyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-l-yloxylpropan-2-amine, (2S)-1- (heptyloxy)-N ,N-dimethyl-3 -R9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxylpropan-2- amine, N,N- dimethyl- l-(nonyloxy)-3-R9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxylpropan-2-amine, N,N-dimethyl- l-[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-l-[(6Z,9Z,12Z)- octadeca-6,9,12-trien-l-yloxy]-3-(octyloxy)propan-2-amine, (2S)-l-[(HZ.14Z)-icosa-ll,14- dien-l-yloxy]-N,N-diniethyl-3-(pentyloxy)propan-2-amine, (2S)-l-(hexyloxy)-3-[(llZ,14Z)- icosa- 11 ,14-dien- 1 -yloxy] -N,N -dimethylpropan-2-amine, 1 - [( 11 Z, 14Z)-icosa- 11 ,14-dien- 1 - yloxy ]-N,N-dimethyl -3-(octyloxy)propan-2-amine, 1 - [( 13Z, 16Z)-docosa- 13,16-dien- 1 -yloxy ]- N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)- 1-[(13Z, 16Z)-docosa- 13,16-dien- 1 -yloxy] -3- (hexyioxy)-N,N -dimethylpropan-2-amine, (2S)- 1 ■■ [( 13Z)-docos- 13-en- 1 -yloxy] -3 ■ (hexyloxy) ■ Attorney Docket No. 11538-008W01

[0192] N,N-dimethylpropan-2-amine, l-[(13Z)-docos-13-en-l-yloxy]-N,N-dimethyl-3- (octyloxy)propan-2-amine, l-[(9Z)-hexadec-9-en-l-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2- amine, (2R)-N,N"dimethyl-H(l"metoyloctyl)oxy]-3-[(9Z,12Z)"Octadeca-9,12-dien- 1- yloxy]propan-2-amine, (2R)-l-[(3,7-dimetbyloctyl)oxyj-N,N-dimethyl-3-R9Z,12Z)-octadeca- 9,12-dien-l-yioxylpropan-2-amine, N,N-dimethyl-l-(octyloxy)-3-((8-RlS,25)-2-([(lR,2R)-2- pentylcyclopropyl]methyljcyclopropyl]octyl }oxy)propan-2-amine, N,N-dimethyl-l-l [8-(2- oclylcyclopropyl)octyl]oxy}-3“(octyloxy)propan-2-amine and (11E,2OZ,23Z)--N,N- dimethylnonacosa-ll,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the lipid may be a cationic lipid such as, but not limited to, DLin- DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid may be the lipids described in and / or made by the methods described in U.S. Patent Publication No. US20130150625, herein incorporated by reference in its entirety. In some embodiments, the nanoparticles described herein may include an amine cationic lipid such as those described in International Patent Application No. WO2013059496, the contents of which are herein incorporated by reference in their entirety. In some embodiments, the cationic lipids may have an amino-amine or an amino-amide moiety.

[0193] In some embodiments, the cationic lipid may be a low molecular weight cationic lipid such as those described in U.S. Patent Application No. 20130090372, the contents of which are herein incorporated by reference in their entirety.

[0194] Ionizable Lipids

[0195] Exemplary ionizable lipids are described in the US patent publications Nos. U.S. 2016 / 0311759. U.S. 2015 / 0376115, U.S. 2016 / 0151284, U.S. 2017 / 0210697, U.S. 2015 / 0140070, U.S. 2013 / 0178541, U.S. 2013 / 0303587, U.S. 2015 / 0141678, U.S. 2015 / 0239926, U.S. 2016 / 0376224, U.S. 2017 / 0119904, U.S. 2012 / 0149894, U.S. 2015 / 0057373, U.S. 2013 / 0090372, U.S. 2013 / 0274523, U.S. 2013 / 0274504, U.S. 2013 / 0274504, U.S. 2009 / 0023673, U.S. 2012 / 0128760, U.S. 2010 / 03241240, U.S. 2014 / 0200257, U.S. 2015 / 0203446, U.S. 2018 / 0005363, U.S. 2014 / 0308304, U.S. 2013 / 0338210, U.S. 2012 / 0101148, U.S. 2012 / 0027796, U.S. 2012 / 0058144, U.S. 2013 / 0323269, U.S. 2011 / 0117125, U.S. 2011 / 0256175, U.S. 2012 / 0202871, U.S. 2011 / 0076335. U.S. 2006 / 0083780, U.S. 2013 / 0123338. U.S. 2015 / 0064242, U.S. 2006 / 0051405, U.S. 2013 / 0065939, U.S. 2006 / 0008910, U.S. 2003 / 0022649, U.S. 2010 / 0130588, U.S. 2013 / 0116307, U.S. 2010 / 0062967, U.S. 2013 / 0202684, U.S. 2014 / 0141070, U.S. 2014 / 0255472, U.S. 2014 / 0039032, U.S. 2018 / 0028664, U.S. Attorney Docket No. 11538-008W01

[0196] 2022 / 0009878, U.S, 2022 / 0040325, U.S. 2012 / 61657480, U.S. 2016 / 0074514, U.S. 2013 / 0330401 , U.S. 2019 / 0185410, U.S. 2012 / 61617468, U.S. 2019 / 0032087, U.S. 2015 / 62184188, U.S. 2019 / 0127318, U.S. 2021 / 0002813, U.S. 2020 / 0345641, U.S. 2014 / 61944336, U.S. 2012 / 61657480, U.S. 2021 / 0059953, U.S. 2022 / 0162521, U.S. 2022 / 0235377, U.S. 2018 / 0085474, U.S. 2018 / 0000953, U.S. 2020 / 0129445, U.S. 2021 / 0145982, U.S. 2021 / 0378980, U.S. 2020 / 0254086, U.S. 2021 / 0346306, and U.S. 2018 / 0000953, the contents of all of which are incorporated herein by reference in their entirety..

[0197] In some embodiments, the nanoparticle comprises an ionizable lipid in a molar ratio of from 0% to 80%. In some embodiments, the ionizable lipid can be present in a molar ratio of at least 0%, (e.g., at least 5%, at least 10%, at least 20%, at least 30%', at least 40%', at least 50%, at least 60%, at least 70%, or at least 80%). In some embodiments, the ionizable lipid can be present in a molar ratio of 80% or less, (e.g., 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1 % or less, or 0.5% or less).

[0198] The ionizable lipid can be present in a molar ratio ranging from any of the minimum values described above to any of the maximum values described above. For example, in some embodiments, the ionizable lipid can be present in a molar ratio of from 0% to 80% (e.g., from greater than 0% to 80%, from greater than 0% to 70%, from greater than 0% to 60%, from greater than 0% to 50%, from greater than 0% to 40%, from greater than 0% to 30%, from greater than 0% to 20%, from greater than 0% to 10%, from greater than 0% to 5%, from greater than 0% to 1%, from greater than 0% to 0.5%, from 1% to 30%, from 1% to 20%, from 1 % to

[0199] 10%, from 1 % to 5%, from 5% to 30%, from 5% to 20%, from 5% to 10%, from 10% to 30%, from 10% to 20%, from 20% to 30%, from 20% to 40%, or from 30% to 40%).

[0200] Helper Lipids

[0201] In some embodiments, the nanoparticle comprises a helper lipid. In some embodiments, the helper lipid can be a non-cationic lipid. In some embodiments, the non-cationic lipid can include, but is not limited to, l,2-dioleoyl-sn-glycero-3"phosphoethanolamine (DOPE), 1- palmitoyl-2-oleoyI-sn-glycero-3-phosphoethanolamine (POPE), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1 -stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1 ,2-dipalmitoyl-sn-glycero-3- phosphocholine), l,2-dioleyl-sn-glycero-3-phosphotidylcholine (DOPC), 1 ,2-dipalmitoyI-sn-glycero-3-phosphoethanoIamine (DPPE), 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine (DMPE), l,2-dioleoyl-5 / 7-glycero-3- phospho-(l’-rac-glycerol) (DOPG), or combinations thereof. In one embodiment, the non-cationic lipid is 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE). In one embodiment, the non-cationic lipid is 1 - palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), In one embodiment, the non- Attorney Docket No. 11538-008W01 cationic lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the non-cationic lipid is l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPH). While several non-cationic lipids are described here, additional non-cationic lipids can be used in combination with the compounds disclosed herein.

[0202] In some embodiments, the nanoparticle comprises a helper lipid in a molar ratio of from 0% to 20%. In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 15%, or 20%.

[0203] Polyeth ylen e Giycol-Lipid

[0204] In some embodiments, the nanoparticle includes a polyethylene glycol-lipid (PEG- lipid). PEG-lipid is incorporated to form a hydrophilic outer layer and stabilize the particles. Nonlimiting examples of polyethylene glycol-lipids include PEG-modified lipids such as PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include DMG-PEG, DLPE-PEGs, DMPE-PEGs, DPPC-PEGs, and DSPE-PEGs. In one embodiment, tire polyethylene glycol-lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG). In one embodiment, the polyethylene glycol-lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-XXXX, wherein XXXX signifies the molecular weight of the polyethylene glycol moiety, e.g. DMG-PEG2000 or DMG-PEG5000.

[0205] In some embodiments, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of from 0% to 5%. In some embodiments, tire nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, or 5%. In one embodiment, the nanoparticle comprises a polyethylene glycol-lipid in a molar ratio of 0.75%.

[0206] In some embodiments, the ratio of PEG in tire lipid nanoparticle formulations may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified from Cl 4 to Cl 8 to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulations. As a non-limiting example, lipid nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and / or 3.0% to 5.0% of the lipid molar ratio of PEG-c-DOMG (R-3-[(®-methoxy-poly(ethyleneglycol)2000)carbamoyl)]- l,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the cationic lipid, DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, Attorney Docket No. 11538-008W01 methoxypoly ethylene glycol), PEG-DMG (1 ,2-Dimyristoyl-sn-glycerol) and / or PEG-DPG (1,2- Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).

[0207] In some embodiments, the LNP formulations may contain PEG-c-DOMG at 3% lipid molar ratio. In some embodiments, the LNP formulations may contain PEG-c-DOMG at 1.5% lipid molar ratio.

[0208] In some embodiments, the pharmaceutical compositions may include at least one of the PEGylated lipids described in International Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.

[0209] In some embodiments, the LNP formulation may contain PEG-DMG 2000 (1 ,2- dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000). In some embodiments, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component. In some embodiments, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see e.g., Gcal! et al., Nonviral delivery of self-amplifying RNA (e.g., mRNA) vaccines, PNAS 2012; PMID: 22908294, the contents of each of which sire herein incorporated by reference in their entirety).

[0210] Sterols

[0211] In some embodiments, the nanoparticle includes a sterol. Sterols are well known to those skilled in the art and generally refers to those compounds having a perhydrocyclopentanophenanthrene ring system and having one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, and the like.

[0212] In some embodiments, the sterol is selected from a cholesterol-based lipid. In some embodiments, the one or more cholesterol -based lipids are selected from cholesterol, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N- ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino- propyl)piperazine, or combinations thereof.

[0213] The sterol can be used to tune the particle permeability and fluidity base on its function in cell membranes. In one embodiment, the sterol is cholesterol.

[0214] In some embodiments, the nanoparticle compri ses a sterol in a molar ratio of from 20% to 80%. In some embodiments, the nanoparticle comprises a sterol in a molar ratio of 25%, 30%, 35%, 40%, 45%, or 50%. In one embodiment, the nanoparticle comprises a sterol in a molar ratio of 40%. Attorney Docket No. 11538-008W01

[0215] The nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a nucleic acid sequence (e.g., mRNA) described herein. As a nonlimiting example, the carbohydrate carrier may include, but is not limited to, an anhydride- modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anbydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication Mo. W02012109121; the contents of which are herein incorporated by reference in their entirety).

[0216] Nanoparticle formulations of the present disclosure may be coated with a surfactant or polymer in order to improve the delivery of the particle. In some embodiments, the nanoparticle may be coated with a hydrophilic coating such as, but not limited to, PEG coatings and / or coatings that have a neutral surface charge. The hydrophilic coatings may help to deliver nanoparticles with larger payloads such as, but not limited to, RNA (e.g., mRNA) within the central nervous system. As a non-limiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in U.S. Patent Publication No. US20130183244, the contents of which are herein incorporated by reference in their entirety.

[0217] In some embodiments, the lipid nanoparticles of the present disclosure may be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in U.S. Patent Publication No. US20130210991, the contents of which are herein incorporated by reference in their entirety.

[0218] In some embodiments, the lipid nanoparticles of the present disclosure may be hydrophobic polymer particles.

[0219] In some embodiments, an immune response may be elicited by delivering a lipid nanoparticle which may inchide a nanospecies, a polymer and an immunogen. (U.S. Publication No. 20120189700 and International Publication No. W02012099805; each of which is herein incorporated by reference in their entirety). The polymer may encapsulate the nanospecies or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, a modified RNA and / or a polynucleotide described herein. In some embodiments, the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.

[0220] Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes). Nanoparticles larger than 10-200 nm which are preferred for Attorney Docket No. 11538-008W01 higher drug encapsulation efficiency and the ability to provide the sustained deli very of a wide array of drags have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosa tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm-500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al. Adv Drag Deliv Rev. 2009 61(2): 158-171; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and / or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No. 8,241,670 or International Patent Publication No. W02013110028, the contents of each of which are herein incorporated by reference in its entirety.

[0221] The lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and / or a polymer-vitamin conjugate and / or a tri-block co-polymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, poly carbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, poly acetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and / or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No. WO2013116804, the contents of which are herein incorporated by reference in their entirety. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (see e.g., International App. No. WO201282165, herein incorporated by reference in its entirety). Nonlimiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (FA'A), poly(lactic acid) (PL. A), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly / lactic acid-co-glycolic acid) (PLGA), polyfL-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D.L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L.-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PEL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, polyfester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene Attorney Docket No. 11538-008W01 glycols such as polyethylene glycol) (PEG), polyalkylene oxides (PEG), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly( vinyl acetate), polyvinyl halides such as poly( vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethyl cellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), polytisopropyl acrylate), poly(lsobutyl acrylate), polyioctadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, polylbutyric acid), poly( valeric acid), poly(lactide-co-caprolactone), PEG- PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer (such as a branched polyether-polyamide block copolymer described in International Publication No. WO2013012476, herein incorporated by reference in its entirety), and (polylethylene glycol))- (poly (propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., U.S. Publication 20120121718 and U.S. Publication 20100003337 and U.S. Pat. No. 8,263,665, the contents of each of which is herein incorporated by reference in their entirety). The co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created. For example, the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new' chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew.

[0222] Chem. Int. Ed. 2011 50:2597-2600; the contents of which are herein incorporated by reference in their entirety). A non-limiting scalable method to produce nanoparticles which can penetrate human mucus is described by Xu et al. (see, e.g., J Control Release 2013, 170(2):279-86; the contents of w Inch are herein incorporated by reference in their entirety ).

[0223] The vitamin of the polymer- vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains). Attorney Docket No. 11538-008W01

[0224] The lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin p4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase. The surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle, (see e.g., U.S. Publication 20100215580 and U.S. Publication 20080166414 and US20130164343; the contents of each of which are herein incorporated by reference in their entirety).

[0225] In some embodiments, the mucus penetrating lipid nanoparticles may comprise at least one polynucleotide described herein. The polynucleotide may be encapsulated in the lipid nanoparticle and / or disposed on the surface of the particle. The polynucleotide may be covalently coupled to the lipid nanoparticle. Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles.

[0226] In some embodiments, the compositions can be formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SEN) may be spherical with an average diameter between 10 to 1000 nm. SEN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and / or emulsifiers. In some embodiments, the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et ah, ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of which are herein incorporated by reference in their entirety). As a non-limiting example, the SEN may be the SEN described in International Patent Publication No. W02013105101, the contents of which are herein incorporated by reference in their entirety. As another non-limiting example, the SEN may be made by the methods or processes described in International Patent Publication No. WO2013105101 , the contents of which are herein incorporated by reference in their entirety.

[0227] In some embodiments, the compositions of the present disclosure may be encapsulated in a nanoparticle. Nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. W02010005740, W02010030763, WO2010005721 , WO2010005723, WO2012054923, U.S. Publication Nos. US20110262491, US20100104645, US20I00087337, US20100068285, US20110274759, US20100068286, Attorney Docket No. 11538-008W01

[0228] 8,293,276, 8,318,208 and 8,318,211; the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, the contents of which are herein incorporated by reference in their entirety.

[0229] In some embodiments, the nanoparticle may be formulated for sustained release. As used herein, “sustained release” refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle may comprise a polymer and nucleic acid sequence of the present disclosure (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, the sustained release formulation may comprise agents which permit persistent bioavailability such as, but not limited to, crystals, macromolecular gels and / or particulate suspensions (see U.S. Patent Publication No US20130150295, the contents of each of which are incorporated herein by reference in their entirety).

[0230] In some embodiments, the nanoparticles of the present disclosure may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide- co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.

[0231] In some embodiments, the nanoparticle comprises a diblock copolymer. In some embodiments, the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, poly acrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester) or combinations thereof. In yet another embodiment, the diblock copolymer may be a high-X diblock copolymer such as those described in International Patent Attorney Docket No. 11538-008W01

[0232] Publication No. WO2013120052, the contents of which are incorporated herein by reference in their entirety.

[0233] As a non-limiting example, the nanoparticle comprises a PLGA-PEG block copolymer (see U.S. Publication No. US20120004293 and U.S. Pat. No. 8,236,330, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968 and International Publication No. WO2012166923, the contents of each of which are herein incorporated by reference in their entirety). In yet another non -limiting example, the nanoparticle is a stealth nanoparticle or a target-specific stealth nanoparticle as described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.

[0234] In some embodiments, the nanoparticle may comprise a multiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Pub. No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).

[0235] In yet another non-limiting example, the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-betal gene delivery vehicle in Lee et al. Thermosensitive Hydrogel as a Tgf-pi Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000; as a controlled gene delivery system in Li et al. Controlled Gene Delivery- System Based on Thermosensitive Biodegradable Hydrogel. Pharmaceutical Research 2003 20(61:884-888; and Chang et al., Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J Controlled Release. 2007 118:245-253, the contents of each of which are herein incorporated by reference in their entirety). The nucleic acid (e.g., mRNA) vaccines of the present disclosure may be formulated in lipid nanoparticles comprising the PEG-PLGA-PEG block copolymer.

[0236] In some embodiments, the nanoparticle may comprise a multiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Pub. No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).

[0237] In some embodiments, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer, (see e.g., U.S. Publication No. 20120076836, the contents of which are herein incorporated by reference in their entirety).

[0238] In some embodiments, the nanoparticle may comprise at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and Attorney Docket No. 11538-008W01 methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly( acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.

[0239] In some embodiments, the nanoparticles may comprise at least one poly( vinyl ester) polymer. The poly( vinyl ester) polymer may be a copolymer such as a random copolymer. As a non-limiting example, the random copolymer may have a structure such as those described in International Application No. WO2013032829 or U.S. Patent Publication No US20130121954, the contents of each of which are herein incorporated by reference in their entirety. In some embodiments, the polyfvinyl ester) polymers may be conjugated to the polynucleotides described herein.

[0240] In some embodiments, the nanoparticle may include at least one diblock copolymer. The diblock copolymer may be, but it not limited to, a poly(lactic) acid-poly(ethylene)glycol copolymer (see, e.g., International Patent Publication No. WO2013044219, the contents of which are herein incorporated by reference in their entirety). As a non-limiting example, the nanoparticle may be used to treat cancer (see International publication No. WO2013044219, the contents of which are herein incorporated by reference in their entirety).

[0241] In some embodiments, the nanoparticles may include at least one cationic polymer described herein and / or known in the art.

[0242] In some embodiments, the nanoparticles may include at least one aniine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poiy(beta-amino esters) (see, e.g., U.S. Pat. No. 8,287,849, the contents of which are herein incorporated by reference in their entirety) and combinations thereof.

[0243] In some embodiments, the nanoparticles may comprise at least one degradable polyester which may contain poiycationic side chains. Degradeable polyesters include, but fire not limited to, polyiserine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[0244] In some embodiments, the compositions may be formulated in colloid nanocarriers as described in U.S. Patent Publication No. US20130197100, the contents of which are herein incorporated by reference in their entirety.

[0245] In some embodiments, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticle may be formulated by the Attorney Docket No. 11538-008W01 methods described in U.S. Publication No. 20120282343, the contents of which are herein incorporated by reference in their entirety.

[0246] In some embodiments, LNPs comprise the lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No. 2012 / 0295832, the contents of which are herein incorporated by reference in their entirety. Activity and / or safety (as measured by examining one or more of ALT / AST, white blood cell count and cytokine induction, for example) of LNP administration may be improved by incorporation of such lipids. LNPs comprising KL52 may be administered intravenously and / or in one or more doses. In some embodiments, administration of LNPs comprising KL52 results in equal or improved mRNA and / or protein expression as compared to LNPs comprising MC3.

[0247] In some embodiments, the lipid nanoparticle may be a limit size lipid nanoparticle described in International Patent Publication No. WO2013059922, the contents of which are herein incorporated by reference in their entirety. The limit size lipid nanoparticle may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; where the lipid bilayer may comprise a phospholipid such as, but not limited to, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, a C8-C20 fatty acid diacylphophatidylcholine, and l-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In some embodiments, the limit size lipid nanoparticle may comprise a polyethylene glycol-lipid such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

[0248] In some embodiments, the compositions may be formulated in a nanoparticle comprising an inner core comprising a non-cellular material and an outer surface comprising a cellular membrane. The cellular membrane may be derived from a cell or a membrane derived from a virus. As a non-limiting example, the nanoparticle may be made by the methods described in International Patent Publication No. WO2013052167, the contents of which Eire herein incorporated by reference in their entirety. As another non-limiting example, the nanoparticle described in International Patent Publication No. WO2013052167, the contents of which are herein incorporated by reference in their entirety, may be used to deliver the compositions described herein.

[0249] In some embodiments, the compositions may be formulated in porous nanoparticle- supported lipid bilayers (protocells). Protocells are described in International Patent Publication No. W02013056132, the contents of which are herein incorporated by reference in their entirety.

[0250] In some embodiments, the compositions described herein may be formulated in polymeric nanoparticles as described in or made by the methods described in U.S. Pat. Nos. Attorney Docket No. 11538-008W01

[0251] 8,420,123 and 8,518,963 and European Patent No. EP2073848B1, the contents of each of which are herein incorporated by reference in their entirety. As a non-limiting example, the polymeric nanoparticle may have a high glass transition temperature such as the nanoparticles described in or nanoparticles made by the methods described in U.S. Pat. No. 8,518,963, the contents of which are herein incorporated by reference in their entirety. As another non -limiting example, the polymer nanoparticle for oral and parenteral formulations may be made by the methods described in European Patent No. EP2073848B1, the contents of which are herein incorporated by reference in their entirety.

[0252] In some embodiments, the compositions described herein may be formulated in nanoparticles used in imaging. The nanoparticles may be liposome nanoparticles such as those described in U.S. Patent Publication No US20130129636, herein incorporated by reference in its entirety. As a non-limiting example, the liposome may comprise gadolinium(III)2-{4,7-bis- carboxymethyl-l()-[(N,N-distearylamidomethyl-N ' -amido-methyl]-l,4,7,10-tetra- azacyclododec- 1-yl} -acetic acid and a neutral, fully saturated phospholipid component (see, e.g., U.S. Patent Publication No US20130129636, the contents of which are herein incorporated by reference in their entirety).

[0253] In some embodiments, the nanoparticles which may be used in the present disclosure are formed by the methods described in U.S. Patent Application No. US2.0130130348, the contents of which are herein incorporated by reference in their entirety.

[0254] In some embodiments, the compositions of the present disclosure may be formulated in a swellable nanoparticle. The swellable nanoparticle may be, but is not limited to, those described in U.S. Pat. No. 8,440,231 , the contents of which are herein incorporated by reference in their entirety.

[0255] The compositions of the present disclosure may be formulated in polyanhydride nanoparticles such as, but not limited to, those described in U.S. Pat. No. 8,449,916, the contents of which are herein incorporated by reference in their entirety.

[0256] The nanoparticles and microparticles of the present disclosure may be geometrically engineered to modulate macrophage and / or the immune response. In some embodiments, the geometrically engineered particles may have varied shapes, sizes and / or surface charges in order to incorporate the polynucleotides of the present disclosure for targeted delivery. Other physical features the geometrically engineering particles may have include, but are not limited to, fenestrations, angled aims, asymmetry and surface roughness, charge which can alter the interactions with cells and tissues. As a non-limiting example, nanoparticles of the present Attorney Docket No. 11538-008W01 disclosure may be made by the methods described in International Publication No

[0257] WO 2013082111, the contents of which are herein incorporated by reference in their entirety.

[0258] In some embodiments, the nanoparticles of tire present disclosure may be water soluble nanoparticles such as, but not limited to, those described in International Publication No. W02013090601, the contents of which are herein incorporated by reference in their entirety. The nanoparticles may be inorganic nanoparticles which have a compact and zwitterionic ligand in order to exhibit good water solubility. The nanoparticles may also have small hydrodynamic diameters (HD), stability with respect to time, pH, and salinity and a low level of non-specific protein binding.

[0259] In some embodiments, the nanoparticles of the present disclosure may be developed by the methods described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.

[0260] In some embodiments, the nanoparticles of the present disclosure are stealth nanoparticles or target-specific stealth nanoparticles such as, but not limited to, those described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety. The nanoparticles of the present disclosure may be made by the methods described in U.S. Patent Publication No. US20130172406, the contents of which are herein incorporated by reference in their entirety.

[0261] In some embodiments, the stealth or target- specific stealth nanoparticles may comprise a polymeric matrix. The polymeric matrix may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polyesters, poly anhydrides, polyethers, polyurethanes, polymethacrylates, polyacrylates, polycyanoacrylates or combinations thereof.

[0262] In some embodiments, the nanoparticle may be a nanoparticle-nucleic acid hybrid structure having a high-density nucleic acid layer. As a non-limiting example, the nanoparticle- nucleic acid hybrid structure may made by the methods described in U.S. Patent Publication No. US20130171646, the contents of which are herein incorporated by reference in their entirety. The nanoparticle may comprise a nucleic acid such as, but not limited to, polynucleotides described herein and / or known in the art.

[0263] At least one of the nanoparticles of the present disclosure may be embedded in the core a nanostructure or coated with a low density porous 3-D structure or coating which is capable of Attorney Docket No. 11538-008W01 carrying or associating with at least one payload within or on the surface of the nanostructure. Non-limiting examples of the nanostructures comprising at least one nanoparticle are described in International Patent Publication No. WO2013123523, the contents of which are herein incorporated by reference in their entirety.

[0264] In some embodiments, compositions may be delivered using smaller LNPs. Such particles may comprise a diameter from below 0.1 pm up to 100 nm such as, but not limited to, less than 0.1 pm, less than 1.0 pm, less than 5 pm, less than 10 pm, less than 15 pm, less than 20 pm, less than 25 pm, less than 30 pm, less than 35 pm, less than 40 pm, less than 50 pm, less than 55 pm, less than 60 pm, less than 65 pm, less than 70 pm, less than 75 pm, less than 80 pm, less than 85 pm, less than 90 pm, less than 95 pm, less than 100 pm, less than 125 pm, less than 150 pm, less than 175 pm, less than 200 pm, less than 225 pm, less than 250 pm, less than 275 pm, less than 300 pm, less than 325 pm, less than 350 pm, less than 375 pm, less than 400 pm, less than 425 pm, less than 450 pm, less than 475 pm, less than 500 pm, less than 525 pm, less than 550 pm, less than 575 pm, less than 600 pm, less than 625 pm, less than 650 pm, less than 675 pm, less than 700 pm, less than 725 pm, less than 750 pm, less than 775 pm, less than 800 pm, less than 825 pm, less than 850 pm, less than 875 pm, less than 900 pm, less than 925 pm, less than 950 pm, less than 975 pm, or less than 1000 pm.

[0265] In some embodiments, compositions may be delivered using smaller LNPs, which may comprise a diameter from 1 nm to 100 nm, from 1 nm to 10 nm, 1 nm to 20 nm, from 1 nm to 30 nm, from 1 nm to 40 nm, from 1 nm to 50 nm, from 1 nm to 60 nm, from 1 nm to 70 nm, from 1 nm to 80 nm, from 1 nm to 90 nm, from 5 nm to from 100 nm, from 5 nm to 10 nm, 5 nm to 20 nm, from 5 nm to 30 nm, from 5 nm to 40 nm, from 5 nm to 50 nm, from 5 nm to 60 nm, from 5 nm to 70 nm, from 5 nm to 80 nm, from 5 nm to 90 nm, from 10 to 50 nm, from 20 to 50 nm, from 30 to 50 nm, from 40 to 50 nm, from 20 to 60 nm, from 30 to 60 nm, from 40 to 60 nm, from 20 to 70 nm, from 30 to 70 nm, from 40 to 70 nm, from 50 to 70 nm, from 60 to 70 nm, from 20 to 80 nm, from 30 to 80 nm, from 40 to 80 nm, from 50 to 80 nm, from 60 to 80 nm, from 20 to 90 nm, from 30 to 90 nm, from 40 to 90 nm, from 50 to 90 nm, from 60 to 90 nm and / or from 70 to 90 nm.

[0266] In some embodiments, such LNPs are synthesized using methods comprising microfluidic mixers. Examples of microfluidic mixers may include, but are not limited to, a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published Attorney Docket No. 11538-008W01

[0267] (Langmuir. 2012. 28:3633-40; Belliveau, N. M. et al.. Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012. I:e37; Chen, D. et al.. Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51 , the contents of each of which are herein incorporated by reference in their entirety). In some embodiments, methods of LNP generation comprising SHM, further comprise the mixing of at least two input streams wherein mixing occurs by micro structure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004 / 0262223 and 2012 / 0276209, the contents of each of which are herein incorporated by reference in their entirety.

[0268] In some embodiments, the compositions of the present disclosure may be formulated in lipid nanoparticles created using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet (UMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany).

[0269] In some embodiments, the compositions of the present disclosure may be formulated in lipid nanoparticles created using microfluidic technology (see, e.g., Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each of which is herein incorporated by reference in its entirety). As a non-limiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (see, e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, the contents of which are herein incorporated by reference in their entirety).

[0270] In some embodiments, the composi tions of the present disclosure may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[0271] In some embodiments, the compositions of the disclosure may be formulated in lipid nanoparticles having a diameter from 10 to 100 nm such as, but not limited to, 10 to 20 nm, 10 to 30 nm, 10 to 40 nm, 10 to 50 nm, 10 to 60 nm, nm, 10 to 80 nm, 10 to 90 nm, 20 to 30 Attorney Docket No. 11538-008W01 nm, 20 to 40 nm, 20 to 50 nm, 20 to 60 nm, 2.0 to 70 nm, 20 to 80 nm, 20 to 90 nm, 2.0 to 100 nm. 30 to 40 nm, 30 to 50 nm, 30 to 60 nm, 30 to 70 nm, 30 to 80 nm, 30 to 90 nm, 30 to 100 nm, 40 to 50 nm, 40 to 60 nm, 40 to 70 nm, 40 to 80 nm, about 40 to about 90 nm, about 40 to

[0272] 100 nm, 50 to 60 nm, 50 to 70 nm, 50 to 80 nm, 50 to 90 nm, 50 to 100 nm, 60 to 70 nm, 60 to

[0273] 80 nm, 60 to 90 nm, 60 to 100 nm, 70 to 80 nm, 70 to 90 nm, 70 to 100 nm, 80 to 90 nm, 80 to 100 nm, and / or 90 to 100 nm.

[0274] In some embodiments, the lipid nanoparticles may have a diameter from 10 to 500 nm.

[0275] In some embodiments, the lipid nanoparticle may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater titan 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[0276] Described herein are also composition including an effective amount of a nanoparticle described herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical compositions can include a pharmaceutically acceptable carrier and a nanoparticle including a single chain (rimer nucleic acid (e.g., mRNA) encoding a first T cell epitope, a p2-microglobulin, and a first MHC heavy chain sequence encapsulated in the nanoparticle. In some aspects, the pharmaceutical composition can include a pharmaceutically acceptable carrier and a lipid nanoparticle including a single chain trimer nucleic acid (e.g., mRNA) encoding a first T cell epitope, a p2-microglobulin, and a first MHC heavy chain sequence encapsulated in the lipid nanoparticle.

[0277] Described herein are also composition including an effective amount of a nanoparticle described herein and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical compositions can include a pharmaceutically acceptable carrier and a nanoparticle including a single chain nucleic acid dimer (e.g., mRNA) encoding a T cell epitope, and a primary MHC heavy chain sequence encapsulated in the nanoparticle. In some aspects, the pharmaceutical composition can include a pharmaceutically acceptable carrier and a lipid nanoparticle including a single chain nucleic acid dimer (e.g., mRNA) encoding a T cell epitope, and a primary MHC heavy chain sequence encapsulated in the lipid nanoparticle.

[0278] Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotides directed protein production as these formulations may be able to increase cell transfection by the RNA (e.g., mRNA) polynucleotide; and / or increase the translation of encoded protein. One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et ah, Mol Ther. 2007 15: 713-720; the Attorney Docket No. 11538-008W01 contents of which are incorporated herein by reference in their entirety). The liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the polynucleotide.

[0279] Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[0280] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, tire effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

[0281] In some embodiments, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from 1 ,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), l,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethyiaminoethyl)-[l,3]-dioxoiane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

[0282] In some embodiments, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-iipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6: 1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441 :111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Attorney Docket No. 11538-008W01

[0283] Hum Gene Ther. 2008 19:125-132; U.S, Patent Publication No US20130122104; all of which are incorporated herein in their entireties). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide. As an example a liposome can contain, but is not limited to, 55%' cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10%' PEG-S-DSG, and 15% l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be l,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2- dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.

[0284] In some embodiments, liposome formulations may comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and / or from about 48.5% cholesterol to about 60% cholesterol. In some embodiments, formulations may comprise a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, formulations may comprise from about 5.0%' to about 10.0% DSPC and / or from about 7.0%' to about 15.0% DSPC.

[0285] In some embodiments, the compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (l,2-dioleoyi-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0286] In some embodiments, the compositions may be formulated in a lipid vesicle, which may have crosslinks between functionalized lipid bilayers.

[0287] In some embodiments, the compositions may be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and / or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and / or polyarginine. In some embodiments, the compositions may be formulated in a lipid-polycation complex, which may further include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE). Attorney Docket No. 11538-008W01

[0288] In some embodiments, the composition can be formulated as a lipoplex, such as, without limitation, the ATUPLEX™ system, the DACC system, the DBTC system and other siRNA- lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids (Aleku et al. Cancer Res. 2008 68:9'788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13: 1360-1370; Gutbier et al., Puim Pharmacol. Ther. 2010 23:334-344; Kaufmann et ah Microvasc Res 2010 80:286-293 Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095- 4100; deFougerolles Hum Gene Ther. 2008 19: 125-132, the contents of each of which are incorporated herein by reference in their entirety).

[0289] In some embodiments, such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18: 1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res

[0290] 2010 80:286-293; Santel et al. Gene Ther 2006 13:1222- 1234; Santel et al. Gene Ther 2006 13:1360-1370; Gutbier et al, Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al. Mol. Ther.

[0291] 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al. Science. 2008 319:627-630: Peer and Lieberman. Gene Ther. 2011 18: 1127-1133, the contents of each of which are incorporated herein by reference in their entirety). One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin- MC3-DMA-based lipid nanoparticle formulations, which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18: 1357-1364, the contents of which are incorporated herein by reference in their entirety). Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N- acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et aL, Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al. Mol Membr Biol. 2010 27:286-298; Patil et al, Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al. Biomacromolecules. 2011 12:2708-2714; Zhao et al. Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al. Mol Ther. 2010 18: 1357-1364; Srinivasan et al. Methods Attorney Docket No. 11538-008W01

[0292] Mo! Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 75 / :497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Then 2010 18:2028-2037; Song et al., Nat Biotechnol. 2.005 23:709-717; Peer et al ., Science. 2008 319:627-630; Peer and Lieberman, Gene Then 2011 18: 1127-1133, the contents of each of which are incorporated herein by reference in their entirety).

[0293] In some embodiments, the composition of the present disclosure can be formulated for controlled release and / or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In some embodiments, the compositions may be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the disclosure, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the disclosure may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the disclosure using fluorescence and / or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the disclosure are encapsulated in the delivery agent.

[0294] In some embodiments, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (U.S. Patent Nos. 9,901,554 and 9,795,679, the contents of each of which are incorporated herein by reference in their entirety).

[0295] In some embodiments, the compositions may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and / or surgical sealant described herein and / or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Attorney Docket No. 11538-008W01

[0296] Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.).

[0297] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery may also include at least one degradable polyester which may contain polycationic side chains. Degradable polyesters include, but are not limited to, polyfserine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L -proline ester), and combinations thereof. In some embodiments, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[0298] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery may also include at least one PEG and / or PEG related polymer derivatives as described in U.S. Pat. No. 8,404,222, the contents of which are incorporated herein by reference in their entirety.

[0299] In some embodiments, the compositions described herein formulated for controlled release delivery may be the controlled release polymer system described in US20130130348, the contents of which are incorporated herein by reference in their entirety.

[0300] In some embodiments, the synthetic nanocarriers may be formulated for targeted release. In some embodiments, the synthetic nanocarrier is formulated to release the polynucleotides at a specified pH and / or after a desired time interval. As a non-limiting example, tire synthetic nanoparticle may be formulated to release the RNA (e.g., mRNA) vaccines after 24 hours and / or at a pH of 4.5 (see International Publication Nos. W02010138193 and W02010138194 and U.S. Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entireties).

[0301] In some embodiments, the synthetic nanocarriers may be formulated for controlled and / or sustained release of the nucleic acids described herein. As a non-limiting example, the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and / or as described in International Pub No. W02010138192 and US Pub No.

[0302] 20100303850, each of which is herein incorporated by reference in their entirety.

[0303] In some embodiments, the synthetic nanocarrier may be formulated for use as a vaccine. As a non-limiting example, the synthetic nanocarrier may include but is not limited to nanocarriers described in International Publication No. W02011150264, W02011150249, WO2012024621 , WO201202629, WO2012024632 and U.S. Publication No. US20110293701, U.S. Publication No. US20110293723, US20120064110, US20120058I53 and US20120058154 the contents of each of which are herein incorporated by reference in their entirety. Attorney Docket No. 11538-008W01

[0304] The vaccine dosage form may be selected by methods described herein, known in the art and / or described in International Publication No, WO2011150258 and U.S. Publication No.

[0305] US20120027806, the contents of each of which arc herein incorporated by reference in their entirety).

[0306] In some embodiments, the compositions may be formulated in colloid nanocarriers as described in U.S. Patent Publication No. US20130197I00, the contents of which are herein incorporated by reference in their entirety.

[0307] In some embodiments, the compositions of the disclosure may be formulated for delivery using the drug encapsulating microspheres described in International Patent Publication No. WO2013063468 or U.S. Pat. No. §,440,614, the contents of each of which are herein incorporated by reference in their entirety. The microspheres may comprise a compound of the formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety. In some embodiments, the amino acid, peptide, polypeptide, lipids (APPL) are useful in delivering the RNA (e.g., mRNA) polynucleotides of the disclosure to cells (see International Patent Publication No. WO2013063468, the contents of which are herein incorporated by reference in their entirety).

[0308] In some embodiments, the compositions may be delivered, localized and / or concentrated in a specific location using the delivery methods described in International Patent Publication No. WO2013063530, the contents of which are herein incorporated by reference in their entirety. As a non-limiting example, a subject may be administered an empty polymeric particle prior to, simultaneously with or after delivering the compositions described herein to the subject. The empty polymeric particle undergoes a change in volume once in contact with the subject and becomes lodged, embedded, immobilized or entrapped at a specific location in the subject.

[0309] In some embodiments, the compositions may be formulated in an active substance release system (see, e.g., U.S. Patent Publication No. US20130102545, the contents of which are herein incorporated by reference in their entirety). The active substance release system may comprise 1) at least one nanoparticle bonded to an oligonucleotide inhibitor strand which is hybridized with a catalytically active nucleic acid and 2) a compound bonded to at least one substrate molecule bonded to a therapeutically active substance (e.g., polynucleotides described herein), where the therapeutically active substance is released by the cleavage of the substrate molecule by the catalytically active nucleic acid.

[0310] In some embodiments the compositions may be associated with a cationic or polycationic compounds, including protamine, nucleoline, spermine or spermidine, or other cationic peptides Attorney Docket No. 11538-008W01 or proteins, such as poly-L-lysine (PEL), polyarginine, basic polypeptides, cell penetrating peptides (CPPs), including HIV-binding peptides, HIV-l Tat (HIV), Tat-derived peptides, Penetratin, VP22 derived or analog peptides, Pestivirus Ems, HSV, VP22 (Herpes simplex), MAP, KAL A or protein transduction domains (PTDs), PpT620, prolin-rich peptides, arginine- rich peptides, lysine -rich peptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(l), pVEC, hCT-derived peptides, SAP, histones, cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA: [l-(2,3-sioleyloxy)propyl)]-N,N,N- trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-ChoI, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol- amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio (propane, DC-6- 14: O,O-ditetradecanoyl-N-.aIpha.-trimethylammonioacetyl)diethanoIamine chloride, CLIP 1 : rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylanimonium chloride, CLIP6: rac- [2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CL1P9: rac-[2(2,3- dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine, or cationic or polycationic polymers, e.g. modified poly aminoacids, such as beta-aminoacid-polymers or reversed polyamides, etc., modified polyethylenes, such as PVP (poly(N-ethyl-4- vinylpyridinium bromide)), etc., modified acrylates, such as pDMAEMA (polyldimethylaminoethyl methylacrylate)), etc, modified amidoamines such as pAMAM (poly(amidoamine)), etc, modified polybetaminoester (PBAE), such as diamine end modified 1,4 butanediol diacrylate-co-5-amino-l -pentanol polymers, etc, dendrimers, such as polypropylamine dendrimers or pAMAM based dendrimers, etc, polyimine(s), such as PEI: poly (ethyleneimine), poly(propyleneimine), etc, polyallylamine, sugar backbone based polymers, such as cyclodextrin based polymers, dextran based polymers, chitosan, etc, silan backbone based polymers, such as PMOXA-PDMS copolymers, etc, blockpolymers consisting of a combination of one or more cationic blocks (e.g. selected from a cationic polymer as mentioned above) and of one or more hydrophilic or hydrophobic blocks (e.g. poly ethylenegly cole) , etc .

[0311] In other embodiments, the composition is not associated with a cationic or polycationic compound. Attorney Docket No. 11538-008W01

[0312] Vaccines

[0313] In some embodiments, the composition can comprise a vaccine. In some embodiments, vaccines can be formulated in a nanoparticle described herein. In some embodiments, vaccines can be formulated in a lipid nanoparticle described herein. In some embodiments, vaccines can be formulated in a lipid-polycation complex described herein, referred to as a cationic lipid nanoparticle. The vaccines of the disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles described herein. In some embodiments, pharmaceutical compositions of vaccines can include liposomes described herein. In some embodiments, the vaccines may be formulated in a lipid vesicle, which may have crosslinks between functionalized lipid bilayers. In some embodiments, the vaccines may be formulated in a lipid-polycation complex.

[0314] Methods of Use

[0315] Described herein are methods of treating or preventing EBV infection in a subject, the methods including: administering to the subject a therapeutically effective amount of the compositions described herein or nucleic acid including SEQ. ID. NO: 4 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity,.

[0316] Described herein are also methods of preventing a disease or disorder caused by EBV infection in a subject, the method including: administering to the subject a therapeutically effective amount of the composition described herein or the nucleic acid including SEQ. ID. NO: 4 or a variant having at least 90% (e.g. 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity,.

[0317] Also described herein are methods of treating a disease or disorder caused by EBV infection in a subject, the method including: administering to the subject a therapeutically effective amount of the composition described herein or the nucleic acid including SEQ. ID. NO: 4 or a variant having at least 90% (e.g. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity,. In some embodiments, the disease or disorder caused by EBV infection is selected from the group consisting of chronic periodontitis, EBV-associated cancer, multiple sclerosis, post-transplantation lymphoproliferative disease, chronic immune dysregulation, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, and combinations thereof. In some embodiments, the EBV-associated cancer is selected from lymphoma, nasopharyngeal carcinoma, and gastric carcinoma.

[0318] Some specific embodiments of composi tions and methods of use include the following: Attorney Docket No. 11538-008W01

[0319] Use of a composition comprising one or more nucleic acids encoding full-length EBV envelope proteins that play key roles in EBV entry and infection of its target cells, namely, EBV gB, gH, gL, gp350 and BMRF2 to provide full protection for both B cells and epithelial cells. Therefore, produced a highly efficient prophylactic vaccine.

[0320] Use of a composition comprising one or more nucleic acids encoding EBV latent proteins and immediate early lytic proteins, namely, LMP1, LMP2, EBNA1, EBNA2. EBNA3A, EBNA3B, EBNA3C and BZLF1 to induce T cell immune responses to eliminate EBV latent infected cells or immediately after lytic acti vation.

[0321] Use of a composition comprising one or more nucleic acids encoding EBV full-length latent proteins LMP1, LMP2, EBNA1, EBNA2, EBN A3 A, EBNA3B, EBNA3C (with deletion / mutation to the key amino acid residues to eliminate potential oncogenic function) and BZLF1 to induce a broad T cell immune response targeting all most all the T cell epitopes contained.

[0322] Use of a composition comprising one or more nucleic acids encoding EBV full-length LMP1, LMP2 (with deletion / mutation to the key amino acid residues to eliminate potential oncogenic function) to induce antibody responses to mediate neutralization, ADCC and ADCP.

[0323] For therapeutic vaccines against the diseases caused by EBV infection, a combination of T cell module and B cell module can be used. The T cell module can comprise one or more nucleic acids encoding for LMP1, LMP2, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C and BZLF1 or a combination thereof (or variants or fragments thereof), and the B cell module can comprise one or more nucleic acids encoding for one or more EBV envelope proteins gB, gH, gL, gp350 and BMRF2 (or variants or fragments thereof).

[0324] In the case of the EBV prophylactic vaccine (with the combination of nucleic acids encoding EBV gB, gH, gL, gp350 and BMRF2), the composition elicits an immune response to EBV envelope proteins that play critical roles in EBV entry and infection of its target cells. This approach completely blocks both pathways of EBV infection of B cells and epithelial cells, and provide protection for both types of target cells, which make the prophylactic vaccine superior to other prophylactic EBV vaccine.

[0325] The combination of T cell module and B cell module can be used to develop therapeutic vaccines for disease caused by EBV infection, based on different mechanisms the disease, part or all the T cell module mRNA vaccine candidates. The T cell module induces T cell immune responses to eliminate latent EBV infected cells or immediate lytic activation, and the B cell module induces antibodies to prevent EBV infection of naive target cells. The T cell module can comprise one or more nucleic acids encoding for an EBV latent protein (e.g., LMP1, LMP2, Attorney Docket No. 11538-008W01

[0326] EBNA1, EBNA2, EBN A3 A, EBNA2B, EBNA3C, or a variant or fragment thereof) and / or immediate early protein BZLF1 or a variant or fragment thereof, and the B cell module can comprise one or more nucleic acids encoding EBV envelope proteins (e.g., gB, gH, gL, gp350 and BMRF2, or a variant or fragment thereof).

[0327] Compositions encoding full-length EBV latent proteins LMP1, LMP2, EBNA1, EBNA2. EBNA3A, EBNA3B, EBNA3C (with deletion / mutation to the key amino acid residues to eliminate potential oncogenic function) and BZLF1 can induce T cell immune responses targeting all most all the T cell epitopes contained. In addition, using compositions encoding EB V full-length LMP1 , LMP2 can also induce antibody responses to mediate neutralization, ADCC and ADCP, which increase further increase efficacy of the vaccine.

[0328] Other example vaccine components include nucleic acids encoding for the polypeptides described in International Publication No. WO2014018858, International Publication No. W02015089340, and International Publication No. WO2018140733, each of which is hereby incorporated by reference in its entirety.

[0329] Methods of A dministra tion

[0330] The compositions described herein can be administered to a subject by any suitable method and technique presently or prospectively known to those skilled in the art to treat or prevent EBV or diseases caused by EBV infection. For example, the active components described herein can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral and parenteral routes of administering. As used herein, the term ‘‘parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the active components of their compositions can be a single administration, or at continuous and distinct intervals as can be readily determined by a person skilled in the art.

[0331] “Excipients” include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and / or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21 st Edition (Lippincott Williams & Wilkins, 2005). Attorney Docket No. 11538-008W01

[0332] Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa buter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. As would be appreciated by one of skill in this art, the excipients may be chosen based on what the composition is useful for. For example, with a pharmaceutical composition, the choice of the excipient will depend on the route of administration, the agent being delivered, time course of delivery of the agent, etc., and can be administered to humans and / or to animals, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), buccally, or as an oral or nasal spray. In some embodiments, the active compounds disclosed herein are administered topically.

[0333] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.

[0334] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone). sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross- linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof. Attorney Docket No. 11538-008W01

[0335] Exemplary surface active agents and / or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly( vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl -pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and / or combinations thereof.

[0336] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0337] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, monothioglycerol, potassium metabisulfite, Attorney Docket No. 11538-008W01 propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0338] Exemplary chelating agents include ethylenediammetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonmm chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and t hi merosal .

[0339] Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0340] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, cblorobutanol, hydroxybenzoate, and phenylethyl alcohol.

[0341] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SEES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germahen II, NeoIone, Kathon, and Euxyl. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0342] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, Attorney Docket No. 11538-008W01 magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen- free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

[0343] Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[0344] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0345] Additionally, the composition may further comprise a polymer. Exemplary polymers contemplated herein include, but are not limited to, cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxy ethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g., the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carageenan, various gums, including xanthan gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac and gum tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic acid and its salts, proteins such as gelatin, collagen, albumin, and fibrin, other polymers, for example, polyhydroxyacids such as polylactide, polyglycolide, polyl(lactide-co- glycolide) and poly(.epsilon.-caprolactone-co-glycolide)-, carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid / acrylamide copolymer, polyalkylene oxides such as polyethylene oxide, Attorney Docket No. 11538-008W01 polypropylene oxide, poly(ethylene oxide- propylene oxide), and a Plutonic polymer, polyoxy ethylene (polyethylene glycol), polyanhydrides, polyvinylalchol, polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG- stearate, l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 1000], l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 2000], and l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000]), copolymers and salts thereof.

[0346] Additionally, the composition may further comprise an emulsifying agent. Exemplary emulsifying agents include, but are not limited to, a polyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses), non-cationic poly(meth)acrylates, non-cationic polyacrylates, such as poly (meth) acrylic acid, and esters amide and hydroxy alkyl amides thereof, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxy vinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. In certain embodiments, the emulsifying agent is cholesterol. Attorney Docket No. 11538-008W01

[0347] Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0348] Injectable compositions, for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be an injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3 -butanediol. Among the acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w / v) sodium carboxymethyl cellulose and 0.1 % (v / v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0349] Composi tions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the particles with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the particles.

[0350] Solid compositions include capsules, tablets, pills, powders, and granules. In such solid compositions, the particles are mixed with at least one excipient and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar- agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution Attorney Docket No. 11538-008W01 retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0351] Tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredlent(s) only, or preferentially, in a certain part of the Intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0352] Compositions for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active compound is admixed with an excipient and any needed preservatives or buffers as may be required.

[0353] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0354] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

[0355] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the nanoparticles in a proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the particles in a polymer matrix or gel.

[0356] The compounds can be incorporated microparticles, nanoparticles, or combinations thereof that provide controlled release of the compounds and / or additional active agents. For Attorney Docket No. 11538-008W01 example, the compounds can be incorporated into polymeric microparticles, which provide controlled release of the drug(s). Release of the drug(s) is controlled by diffusion of the drug(s) out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0357] Polymers, which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, may also be suitable as materials for drug containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0358] Alternatively, the compound can be incorporated into microparticles prepared from materials which tire insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading within the GI tract by means including enzymatic degradation, surfactant action of bile acids, and / or mechanical erosion. As used herein, the term “slowly soluble in water” refers to materials that are not dissolved in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats. Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax. As used herein, a wax-like material is defined as any material, which is normally solid at room temperature and has a melting point of from about 30 to 300° C.

[0359] In some cases, it may be desirable to alter the rate of water penetration into the microparticles. To this end, rate-controlling (wicking) agents may be formulated along with the fats or waxes listed above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried corn starch), cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethyl- Attorney Docket No. 11538-008W01 cellulose), alginic acid, lactose and talc. Additionally, a pharmaceutically acceptable surfactant (for example, lecithin) may be added to facilitate the degradation of such microparticles.

[0360] Proteins, which are water insoluble, such as zein, can also be used as materials for the formation of drug containing microparticles. Additionally, proteins, polysaccharides and combinations thereof, which tire water-soluble, can be formulated with drug into microparticles and subsequently cross-linked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and subsequently cross-linked.

[0361] Encapsulation or incorporation of drug into carrier materials to produce drug-containing microparticles can be achieved through known pharmaceutical formulation techniques. In the case of formulation in fats, waxes or wax-like materials, the carrier material is typically heated above its melting temperature and the drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof. Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion. In a preferred process, wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads. These processes tire known in the art.

[0362] For some carrier materials it may be desirable to use a solvent evaporation technique to produce drag-containing microparticles. In this case drug and carrier material are co-dissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.

[0363] In some embodiments, drug(s) in a particulate form is homogeneously dispersed in a water-insoluble or slowly water soluble material. To minimize the size of the drug particles within the composition, the drug powder itself may be milled to generate fine particles prior to formulation. The process of jet milling, known in the pharmaceutical art, can be used for this purpose. In some embodiments, drug in a particulate form is homogeneously dispersed in a wax or wax like substance by heating the wax or wax like substance above its melting point and adding the drug particles while stirring the mixture. In this case a pharmaceutically acceptable sutfactant may be added to the mixture to facilitate the dispersion of the drug particles.

[0364] The particles can also be coated with one or more modified release coatings. Solid esters of fatty acids, which are hydrolyzed by lipases, can be spray coated onto microparticles or drug particles. Zein is an example of a naturally water-insoluble protein. It can be coated onto drug Attorney Docket No. 11538-008W01 containing microparticles or drug particles by spray coating or by wet granulation techniques. In addition to naturally water-insoluble materials, some substrates of digestive enzymes can be treated with cross-linking procedures, resulting in tire formation of non-soluble networks. Many methods of cross-linking proteins, initiated by both chemical and physical means, have been reported. One of the most common methods to obtain cross-linking is the use of chemical crosslinking agents. Examples of chemical cross-linking agents include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these crosslinking agents, oxidized and native sugars have been used to cross-link gelatin. Cross-linking can also be accomplished using enzymatic means; tor example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products. Finally, cross-linking can be initiated by physical means such as thermal treatment, UV irradiation and gamma irradiation.

[0365] To produce a coating layer of cross-linked protein surrounding drug containing microparticles or drug particles, a water-soluble protein can be spray coated onto the microparticles and subsequently cross-linked by the one of the methods described above. Alternatively, drug-containing microparticles can be microencapsulated within protein by coacervation-phase separation (for example, by the addition of salts) and subsequently crosslinked. Some suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0366] Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations, which cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed: pectin and chitosan, for example, can be complexed via electrostatic interactions.

[0367] In certain embodiments, it may be desirable to provide continuous delivery of one or more compounds to a patient in need thereof. For intravenous or intraarterial routes, this can be accomplished using drip systems, such as by intravenous administration. For topical applications, repeated application can be done or a patch can be used to provide continuous administration of the compounds over an extended period of time.

[0368] The compounds described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymeric implants. In one embodiment, the compounds are incorporated into a polymer that is a liquid or paste at room temperature, but upon contact with aqueous medium, such as physiological fluids, exhibits an increase in viscosity to form a semisolid or solid material. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from the copolymerization of at least one unsaturated hydroxy fatty acid Attorney Docket No. 11538-008W01 copolymerized with hydroxyalkanoic acids. The polymer can be melted, mixed with the active substance and cast or injection molded into a device. Such melt fabrication require polymers having a melting point that is below the temperature at which the substance to be delivered and polymer degrade or become reactive. The device can also be prepared by solvent casting where the polymer is dissolved in a solvent and the drug dissolved or dispersed in the polymer solution and the solvent is then evaporated. Solvent processes require that the polymer be soluble in organic solvents. Another method is compression molding of a mixed powder of the polymer and the drug or polymer particles loaded with the acti ve agent.

[0369] Alternatively, the compounds can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is a solid at room temperature. For example, the compounds can be incorporated into a biodegradable polymer, such as polyanhydrides, polyh ydroalkanoic acids (PHAs), PLA, PGA, P1..GA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin, and combinations thereof and compressed into solid device, such as disks, wafers, or extruded into a device, such as rods.

[0370] The release of the compounds from the implant can be varied by selection of the polymer, the molecular weight of the polymer, and / or modification of the polymer to increase degradation, such as the formation of pores and / or incorporation of hydrolyzable linkages. Methods for modifying the properties of biodegradable polymers to vary the release profile of the compounds from the implant are well known in the art.

[0371] In some embodiments, the compounds or pharmaceutical compositions can be administered locally. In some embodiments, the compounds are incorporated in a delivery system such as gels, nanoparticles, microparticles, or implants such as (e.g., rods, discs, wafers, orthopedic implants) for sustained release. In some embodiments, the compounds can be administered using a local delivery implantable system comprising the compounds incorporated within a gel, nanoparticles, microparticles, or an implant. In some embodiments, the pharmaceutical compositions comprise a delivery system such as gels, nanoparticles, microparticles, or implants such as (e.g., rods, discs, wafers, orthopedic implants) for sustained release of paroxetine or a pharmaceutically acceptable salt or derivative thereof.

[0372] The active ingredient may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular active ingredient, its mode of administration, its mode of activity, and the like. The active ingredient, whether the active compound itself, or the active Attorney Docket No. 11538-008W01 compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within die scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

[0373] The active ingredient may be administered by any route. In some embodiments, the acti ve ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and / or drops), mucosal, nasal, bucal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.

[0374] The exact amount of an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severi ty of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0375] Useful dosages of the compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.

[0376] The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms or disorder are affected. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be Attorney Docket No. 11538-008W01 adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.

[0377] Compositions described herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of compositions may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0378] In some embodiments, the compositions may be administered at dosage levels sufficient to deliver 0.0001 mg / kg to 100 mg / kg, 0.001 mg / kg to 0.05 mg / kg, 0.005 mg / kg to 0.05 mg / kg, 0.001 mg / kg to 0.005 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, or 1 mg / kg to 25 mg / kg, of subject body weight per day, one or more times a day, per week, per month, etc. to obtain the desired therapeutic and / or prophylactic effect (see, e.g., the range of unit doses described in International Publication No WO2013078199, the contents of which are herein incorporated by reference in their entirety). The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three months, every 6 months, etc. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. In exemplary embodiments, compositions may be administered at dosage levels sufficient to deliver 0.0005 mg / kg to 0.01 mg / kg, e.g., about 0.0005 mg / kg to about 0.0075 mg / kg, e.g., about 0.0005 mg / kg, about 0.001 mg / kg, about 0.002 mg / kg, about 0.003 mg / kg, about 0.004 mg / kg or about 0.005 mg / kg.

[0379] In some embodiments, the compositions may be administered once or twice (or more) at dosage levels sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg. Attorney Docket No. 11538-008W01

[0380] In some embodiments, the compositions may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.52.5 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages and frequency of administration are encompassed by the present disclosure. For example, a composition described herein may be administered three or four times.

[0381] In some embodiments, the compositions may be administered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 12.0, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 years later) at a total dose of or at dosage levels sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.

[0382] In some embodiments, the composition (e.g., vaccine) for use in a method of vaccinating a subject is administered to the subject as a single dosage of between 10 ug / kg and 400 pg / kg of the nucleic acid vaccine (in an effective amount to vaccinate the subject). In some embodiments, the composition (e.g., vaccine) for use in a method of vaccinating a subject is administered to the subject as a single dosage of between 10 pg and 400 pg of the nucleic acid vaccine (in an effective amount to vaccinate the subject). In some embodiments, a composition (e.g., vaccine) for use in a method of vaccinating a subject is administered to the subject as a single dosage of 25-1000 pg (e.g., a single dosage of each nucleic acid present in the composition). In some embodiments, a composition (e.g., vaccine) is administered to the subject as a single dosage of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 pg. For example, a composition (e.g., vaccine) may be administered to a subject as a single dose of 25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000, 250-500, 250- 1000, or 500-1000 pg. In some embodiments, a composition (e.g., vaccine) for use in a method Attorney Docket No. 11538-008W01 of vaccinating a subject is administered to the subject as two dosages, the combination of which equals 25-1000 p.g of the composition (e.g., vaccine).

[0383] A composition (e.g., vaccine pharmaceutical composition) described herein can be formulated into a dosage form described herein, such as an intranasal , intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0384] EXAMPLES

[0385] Epstein-Barr virus (EBV) is a gamma human herpesvirus, it is the primary cause of infectious mononucleosis, and a recent epidemiology study showed evidence that EBV is also the cause of multiple sclerosis. EBV is also the first human tumor virus discovered and is strongly implicated in the etiology of multiple lymphoid and epithelial cancers. Additional studies demonstrated that EBV plays key roles in the pathogenesis of other diseases and disorders, including chronic periodontitis, rheumatoid arthritis, and systemic lupus erythematosus.

[0386] EBV envelope proteins including gH / gL, gB, and gp350 play key roles in EBV entry and infection of target cells, and neutralizing antibodies elicited by each of these proteins can prevent EBV infection of target cells and markedly decrease EBV titers in the peripheral blood of humanized mice challenged with lethal dose EBV. Immunization with a combination of gH / gL, gB, and / or gp350 can induce markedly increased synergistic EBV neutralizing activity as compared to immunization with individual proteins. These findings suggest that an EBV prophylactic vaccine that induces neutralizing antibodies holds great promise for prevention of EBV associated diseases.

[0387] Therapeutic EBV vaccines have also been tested clinically with encouraging results. Immunization with various vaccine platforms expressing the EBV latent proteins EBNA1, LMP1, and / or LMP2 promoted specific CD4+ and CD8+ cytotoxic responses with anti-tumor activity.

[0388] The nucleic acid vaccine compositions are described that can be administered both prophylactically to prevent EBV infection as well therapeutically to treat EBV infection as well as diseases caused by EBV, such as multiple sclerosis, EBV associated cancers, chronic immune dysregulation, chronic periodontitis, etc.

[0389] Epstein-Barr virus (EBV) is a gamma human herpesvirus, and has a 170 kb linear, double-stranded DNA genome that encodes more than 80 proteins and 46 functional small untranslated RNAs. EBV primarily infect nasopharyngeal epithelial cells and B cells, and EBV Attorney Docket No. 11538-008W01 infection in epithelial cells usually results in lytic infection, whereas EBV infection in B cells establishes lifelong latent infection.

[0390] EBV infection of epithelial cells initiates with EBV BMRF2 binding to integrins, followed by gH / gL binding to integrins and ephrin receptor A2, triggering activation of gB and caused the fusion of the viral envelope to the plasma membrane of the epithelial cell and EBV entry and infection of epithelial cells. EBV infection of B cells is initiated by attachment to the B cells with the EBV envelope protein gp350 binding to CD21 (complement receptor 2, CR2). EBV gp42 then binds to MHC-II on the B cell surface, leading to its association with the heterodimeric protein gH / gL. EBV gH / gL activates the EBV fusion protein gB, which mediates viral envelope-host cell endosomal membrane fusion, and results in EBV entry and infection of the B cells. Thus, EBV envelope proteins gH / gL and gB are essential for EBV infection of both B cells and epithelial cells, whereas gp350 and BMRF2 are important for efficient infection of B cells and epithelial cells respectively.

[0391] Once infecting the host, EBV establishes two alternative modes of infection: lytic and latent. During the lytic infection, EBV expresses more than 80 lytic proteins, and these proteins facilitate the generation of new EBV viral particles and engage in immune evasion. During the process of developing latency, EBV progresses through three different EBV latency programs characterized by a set of gradually restricted viral gene expression patterns, but no production of EBV virions. Through this process EBV develops eventual lifetime persistence in memory B cells. Latent EBV infections play key roles in the pathogenesis of the diseases caused by EBV infection, such as EBV associated cancer, multiple sclerosis and chronic periodontitis. In type III latency, all eight EBV latent antigens are expressed, including six EBV nuclear' antigens (EBNA 1, EBNA2, EBNA3A, EBNA3B, EBNA 3C and EBNA 6 / LP), and two latent membrane proteins (LMP1 and LMP2). Latency III is mainly seen in PTLD, immunoblastic lymphomas, and most likely in multiple sclerosis. Latency II exists in HL, NPC, GC and likely chronic periodontitis, where EBNA1, LMP1 and LMP2 are expressed. In type I latency, only EBNA1 is expressed and is seen in BL.

[0392] Primary EBV infection in young children usually produces no significant symptoms, whereas primary EBV infection in adolescents and young adults could cause infectious mononucleosis (IM), and up to 70% of adolescents and young adults present with the classical symptoms of IM after EBV infection. IM is characterized by two to four weeks of fever, pharyngitis, cervical lymphadenopathy and fatigue accompanied by a massive expansion of the number of EBV specific CD8+ T cells after an incubation period of about six weeks. The vast majority of IM cases are self-limiting with an excellent prognosis, with rare cases of severe acute Attorney Docket No. 11538-008W01 complications such as splenic rupture, hepatitis and airway obstruction due to tonsil enlargement. Late complications to IM include Hodgkin lymphoma and multiple sclerosis. Life-long latent EBV infection establishes following primary infection.

[0393] EBV is also the first human tumor virus identified, and it is strongly associated with epithelial cell cancers such as nasopharyngeal cancer (NPC), gastric cancer as well as lymphoid cancers such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL) and post transplantation lymphoproliferative disorder (PTLD). NPC is endemic in southeast Asia, and the vast majority are the nonkeratinized type, accounting for 80,000 new eases each year worldwide. Nonkeratinized NPC displays a lymphoepithelial-like (LEL) appearance with a marked lymphocytic infiltration, which is 100% EBV positive. About 10% of gastric cancers are associated with EBV infection, have a similar LEL pathological change and are EBV positive, accounting for about 83,000 new cases each year. Essentially all Burkitt lymphoma in equatorial Africa and in Papua New Guinea, are EBV genome-positive, accounting for 7,000 new cases each year. Hodgkin lymphoma is also strongly associated with EBV, especially the mixed cellularity subtype, of which 80-90% are EBV positive. PTLD is another example that EBV plays a critical role in cancer pathogenesis, all the cases of PTLD are EBV positive, and adoptive transfer of EBV specific T cells could prevent or cure the disease. The role of EBV in cancer pathogenesis has also been confirmed in animal models, as inoculation of cotton top tamarins or humanized mice with high titers of EBV results in the development of B-cell lymphomas and lymphoproliferative disease that are seen in humans.

[0394] Multiple sclerosis (MS) is the most common inflammatory demyelinating disease, affecting both the brain and spinal cord. It is a lifetime, potentially debilitating condition with both remitting / relapsing and progressive phases. It typically occurs in young adults, especially Caucasians with a prevalence of 1 / 1,000 in this latter population and affects -2.5 million people worldwide. EBV has been recognized as the strongest infectious risk factor for MS, etiological evidence including a high titer of anti-EBNAl antibodies observed several years before MS onset, preliminary success of EBV specific T cell therapy for treating progressive MS, and intriguing data from animal modeling. Recent epidemiology study showed EBV infection increased MS 32-fold, and established the etiological role of EBV infection in the pathogenesis of MS. Chronic immune dysregulation (and associated disorders, such as chronic periodontitis) have also been linked to EBV infection.

[0395] There is currently no prophylactic EBV vaccine or therapeutic vaccine for the diseases caused by EBV infection. A phase II clinical trial conducted in EBV seronegative adults using a recombinant monomeric gp350 protein showed 78% efficacy in preventing infectious Attorney Docket No. 11538-008W01 mononucleosis, though the vaccine did not prevent asymptomatic EBV infection. Therapeutic EBV vaccine phase I clinical trials with various vaccine platforms expressing the EBV latent proteins EBNA1, LMP1, and / or LMP2 have also showed encouraging results in the treatment of EBV associated cancers. EBV prophylactic mRNA vaccine and therapeutic mRNA vaccine for the diseases caused by EBV infection are described in this invention disclosure.

[0396] EBV gH / gL and trimeric gB recombinant proteins and nanoparticles have been reported to induce high titers of EBV neutralizing antibodies, and both tire gH / gL antibodies and gB antibodies protected humanized mice from lethal EBV challenge and prevented lymphoma. In a phase 2 clinical trial, a recombinant gp350 protein demonstrated 78% efficacy in preventing infectious mononucleosis, though it did not prevent asymptomatic EBV infection (27, 28). All these data indicated that prevention of EBV entry into its target cells would most likely be an approach to develop an efficient EBV prophylactic vaccine. The combination of EBV envelope proteins that play roles in EBV infection, gB, gH / gL, gp350 and BMRF2, will be used to induce antibodies to prevent EBV infection of both target cells, B cells and epithelial cells. In addition, the T cell immune responses elicited by these EBV envelope proteins will further increase the efficacy.

[0397] A nucleic acid (e.g., mRNA (vaccine platform, and the SARS Cov-2 spike protein vaccine candidate demonstrated > 10-fold higher efficacy than other mRNA vaccines) was successfully developed. This vaccine platform will be used to produce EBV envelope protein vaccine candidates encoding gB, gH / gL, gp350, and / or BMRF2, and use the combination of these mRNAs as a EBV prophylactic vaccine. The proposed EBV prophylactic vaccine will produce EBV envelope proteins that are identical to or closely mimic the native conformation of corresponding EBV envelope proteins, which will elicit even higher neutralizing antibodies compared to their recombinant protein counterparts. The nucleic acid vaccine could also produce envelope proteins that have multiple transmembrane domains such as BMRF2, which is impossible for recombinant protein vaccine candidates.

[0398] For therapeutic vaccines for diseases caused by EBV infection, such as EBV associated cancers, multiple sclerosis, chronic periodontitis, rheumatoid arthritis etc., the nucleic acid vaccine platform will use to express full-length EBV latent proteins LMP1, LMP2, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C as well as lytic protein full-length BZLF1 to induce strong CD4 and CD8 T cell immune response. These strong T cell immune responses especially CDS T cell immune response will help eliminate the latent EBV infected cells and the cells soon after early EBV reactivation. LMP1 and LMP2 would also induce antibody immune responses to mediate neutralization, antibody dependent cytotoxicity (ADCC) and antibody dependent cell Attorney Docket No. 11538-008W01 phagocytosis (ADCP) to further increase the efficacy of the EBV therapeutic vaccine. Depending on the target disease, different combination of T cell antigens can be selected, for example, LMP1, LMP2 and EBNA1 are selected for EBV associated nasopharyngeal carcinoma and lymphoma, LMP1 , LMP2 and BZLF1 are selected for chronic periodontitis, whereas a full set of T cell antigens described above will be selected for multiple sclerosis and post-transplant lymphoproliferative disease. For the EBV latent proteins that induce transformation, deletion and / or mutation to key amino acid residues are made to eliminate the potential oncogenic functions.

[0399] Since lytic EBV infection and EBV replication also play critical roles in the pathogenesis and progression of the diseases caused by EBV infection, a second set of antigens targeting the prevention of EBV entry into its target cells, the components of EBV envelope proteins used in prophylactic vaccine are also included to further increase the efficacy of therapeutic vaccines for the diseases caused by EBV infection. In other words, the therapeutic vaccines for the diseases caused by EBV infection has a T cell module and a B cell module. The T cell module can comprise one or more nucleic acids encoding EBV latent proteins LMP1, LMP2, EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C and immediate early lytic protein BZLF1, and B cell module can comprise one or more nucleic acids encoding EB V envelope proteins gB, gH / gL, gp350 and / or BMRF2.

[0400] Example Vaccine Compositions

[0401] EBV envelope proteins gB, gH, gL, gp350 and BMRF2 play key roles in EBV entry and infection of its target cells, using a combination of mRNA vaccine candidates encoding these EBV envelope proteins to elicit EBV neutralizing antibodies to block EBV entry and protect both B cells and epithelial cells from EBV infection.

[0402] DNA encoding EBV envelope proteins gB, gH, gL, gp350 and BMRF2 are synthesized, cloned into IVS plasmid and IVS is performed to produce mRNA vaccine candidates encoding these EBV envelope proteins. In vitro potency analysis is performed to verify protein expression, mice are immunized with individual or combination of mRNA vaccines encoding these envelope proteins for immunogenicity studies (gB + gH / gL, gB + gH / gL+ gp350 + BMRF2), total (binding) antibodies Eire analyzed by ELISA and EBV neutralizing antibodies are analyzed by flow cytometry.

[0403] Mouse IgG is purified from mice immunized with these EBV envelope proteins individually or in combination, and passive immunization protection from lethal EBV challenge are performed using humanized NSG mice. Attorney Docket No. 11538-008W01

[0404] Prophylactic EBV vaccine can be used to prevent EBV infection as well as the diseases caused by EBV infection, including infectious mononucleosis, multiple sclerosis, chronic periodontitis, EBV associated cancers, rheumatoid arthritis, systemic lupus erythematosus, and others.

[0405] Post transplantation lymphoproliferative disease is a type III latent EBV infection, with all the EBV latent proteins expressed. Both latent EBV infection and EBV replication play critical roles in progression of post transplantation lymphoproliferative disease. The therapeutic vaccine for post transplantation lymphoproliferative disease has a T cell module and a B cell module, the T cell module consist of mRNAs encoding EBV latent protein LMP1 , LMP2, EBNA1, EBNA2, EBNA3A, EBNA2B, EBNA3C and immediate early protein BZLF1, and the B cell module consists of identical mRNAs as that of EBV prophylactic vaccine. Immunization with the combination of T cell module and B cell module will induce potent T cell immune response and strong antibody response. The T cell response will eliminate the latent EBV infected cells, whereas antibodies will prevent EBV infection of naive target cells.

[0406] Although the latent EBV infection in multiple sclerosis has not been officially classified, studies from autopsy of patient brain samples suggest that the latent EBV infection in multiple sclerosis is type Ill, as all the EBV latent proteins are expressed. Thus, the same therapeutic vaccine for post transplantation lymphoproliferati ve disease will be developed for multiple sclerosis.

[0407] For T cell module, DNA encoding EBV latent protein LMP1 , LMP2, EBNA1 , EBNA2, EBNA3A, EBNA2B, EBNA3C and immediate early protein BZLF1 are synthesized, cloned into IVS plasmid and IVS is performed to produce mRNA vaccine candidates encoding these EBV proteins. In vitro potency analysis is performed to verify protein expression, mice are immunized with individual or combination of mRNA vaccines encoding these proteins for immunogenicity studies, CD4 and CD8 T cell immune responses and cytokine production are analyzed by ELISpot or flow cytometry.

[0408] B cell module is identical to that of prophylactic EBV vaccine, which consists of the combination of mRNA vaccine candidates encoding EBV envelope proteins gB, gH, gL, gp350 and BMRF2.

[0409] Active immunization and EBV challenge studies are conducted using humanized DRAGA mice. Humanized DRAGA mice are immunized with the combination of EBV B cell module and T cell module, and EBV challenge studies will be performed to evaluate the efficacy of the vaccines. Attorney Docket No. 11538-008W01

[0410] Latent EBV infection play critical roles in the pathogenesis of EBV associated nasopharyngeal carcinoma and lymphoma, EBNA1, LMP1 and LMP2 are expressed by these cancer cells, and these EBV latent proteins can be used as targets of immune responses. EBV replication also play key roles in progression and metastasis of EBV associated nasopharyngeal carcinoma and lymphoma. Therefore, the therapeutic vaccines for EBV associated nasopharyngeal carcinoma also consists of a T cell module and a B cell module. The T cell module consists of the combination of mRNA vaccine candidates encoding EBNA1, LMP1 and LMP2, and the B cell module is identical to the EBV prophylactic vaccine, which consists of the combination of mRNA vaccine candidates encoding EBV envelope proteins gB, gH, gL, gp350 and BMRF2.

[0411] Immunogenicity of the therapeutic vaccine consists of EBV B cell module in combination with EBNA1, LMP1, LMP2 and BZ.LF1 will be evaluated in mice, and anti-tumor efficacy will be evaluated in humanized mouse modules, especially humanized DRAGA mice. Clinical trials will be conducted to evaluate the efficacy.

[0412] Latent EBV infection and reactivation of lytic EBV infection play key roles in chronic periodontitis, high level LMP1, LMP2 and BZLF1 are expressed in junctional epithelial cells. Therapeutic vaccine for Chronic periodontitis consists of EBV B cell module in combination with LMP1, LMP2 and BZLF1. The elicited immune response will eliminate the latent EBV infected cells and prevent EBV infection of new target cells.

[0413] Individual mRNA vaccine candidate or combination of vaccine candidates are tested for in vitro potency to confirm protein expression, followed by immunization studies in mice to characterize immunogenicity. Clinical trials will be conducted to evaluate the efficacy of EBV therapeutic vaccine for Chronic periodontitis. These vaccine compositions Rheumatoid arthritis, systemic lupus erythematosus, etc.

[0414] Example nucleotide sequences suitable for the preparation of vaccines described herein are included in the table below. Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01 Attorney Docket No. 11538-008W01

[0415] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which Eire illustrated in the accompanying Examples and Figures.

[0416] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the arc that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically related may be substituted for the agents described herein while the same or similar' results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0417] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0418] EXAMPLES

[0419] Example 1: EBV gp350 mRNA LNP vaccine candidates formulated with ARV-T1 induced markedly high titers of neutralizing antibodies and strong T cell immune response in BALB / c mice

[0420] EBV has 2 groups of target cells, B cells and the oral-pharyngeal epithelial cells. EBV gp350 plays a critical role in infection of B cells, as the infection is initiated with EBV gp350 binding to CD21 on B cell surface and more than 50% of the EBV neutralizing antibodies in human sera is induced by EBV gp350. EBV gp350 is a critical vaccine candidate for the Attorney Docket No. 11538-008W01 prevention of EB V infection as well as the diseases caused by EBV infection. 2 EBV gp350 mRNA LNPs were produced and characterized the immunogenicity of these vaccine candidates in BALB / c mice.

[0421] EBV gp350 coding sequence was downloaded from Gene Bank, reference # NC_007605.1. DNA encoding EBV full-length gp350 (gp350FL) and gp350 / 470tetra (gp470T) with flanking 5’-UTR were codon optimized and synthesized, and cloned into pUC57 vector that provided a 3’-UTR and poly A tail downstream. EBV gp470T encoding 2 copies of the first 470 amino acid residues of gp350 in tandem followed by a leucine zipper dimerization domain. EBV mRNA encoding EBV gp350FL or gp470T was produced by in vitro transcription using an optimized T7 RNApolymerase-mediated transcription reaction with uridine-5’ -triphosphate replaced by N1 ■■methylpseudouridine -5 ’■■triphosphate, and a cap 1 structure was added enzymatically using vaccinia capping enzyme and 2’0-methyltransferase.

[0422] EBV gp350 lipid nanoparticle (LNP) formulations were prepared by mixing lipids in an organic phase (ethanol) with mRNA in an aqueous phase (50 mM citrate buffer, pH 4.0), maintaining a specific ratio (1:3, v / v) between the organic and aqueous phases. The total lipid concentration was 12.5 mM, with the molar percentage ratio for constituent lipids as: 50% ATV- T1 (ionizable lipids), 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG in ethanol. All formulations maintained a nitrogen-to-phosphate ratio (N / P) of 4.

[0423] HEK-293T cells were transfected with LNP-mRNA encoding EBV gp350FL or gp470T formulated with ARV-T1, and the expression of gp350 protein was evaluated by Flow cytometry analysis using an EBV gp350 antibody (72A1). Transfection of 293T cells by either gp350FL LNP or mixture of gp350FL mRNA with lipofectamine MessagerMax demonstrated >98% gp350 positive cells as shown by flow cytometer analysis (Figure 1 A and IB). EBV gp470T LNP or mixture of gp470T mRNA with lipofectamine MessagerMax also showed high level gp350 expression, though slightly lower than that of gp350FL (Figure 1A and IB).

[0424] For immunogenicity studies, BALB / c mice were immunized intramuscularly (quadriceps) with LNPs encapsulating gp350FL or gp470T formulated with ARV-T1 on day 1 (prime) and day 21 (boost), and PBS was injected as negative control. Serum was collected two weeks after prime and boost for binding antibody and EBV neutralizing antibody analysis. On day 35, mice were euthanized, and spleens were removed for IFN-y expression study with ELISpot analysis.

[0425] Immunization with gp350FL LNP induced high titers of gp350-specific binding antibodies (104to 105) 21 days after 1stimmunization, and demonstrated clear' dose response (Figure 1C). Compared to gp350FL LNP, immunization with gp470T LNP induced about 10-fold Attorney Docket No. 11538-008W01 lower gp35O-specific binding antibodies 21 days after 1stimmunization (Figure 1C). The titers of gp350 binding antibodies increased at least 10-fold 35 days after 1stimmunization for both gp350FL LNP and gp470T LNP as compared to their corresponding titers elicited 21 days after 1stimmunization. Gp350FL LNP induced 5- to 10-fold higher gp350 binding antibodies compared to gp470 LNP 35 days after 1stimmunization, although only the highest dose showed statistical difference (Figure 1C).

[0426] EB V-neutralizing antibody titers were determined in vitro using AKATA cells (EBV- negative human Burkitt lymphoma cell line) and EBV-GFP derived from CNE2 cells. Immunization with gp350FL LNP induced high titers of EBV neutralizing antibodies, in the range of IO4to 10s, and showed a trend of dose response (Figure ID). Gp470 LNP induced slightly lower titers of EBV neutralizing antibodies, and were not statistically significant compared to that of gp350FL LNP (Figure ID). Currently Dr. Jeffrey Cohen group at NIH reported the highest titer of EBV neutralizing antibodies elicited by gp350 nanoparticles, after immunization 3 times with 5 pg gp350 nanoparticles, the titers of B cell EBV neutralizing antibodies was about -6,000 (Figure IE). In contrast, the titers of B cell EBV neutralizing antibodies induced by our gp350FL LNP was about -200,000 after immunization with 5 pg LNP, which was more than 30-fold higher than that of gp350 nanoparticles (Figure ID and IE). Although the EBV neutralization analyses were performed by different labs using different EBV strains, literature showed that the results could be compared, as most of the labs used a similar method, and the analysis of human sera showed similar EBV neutralizing titers.

[0427] Mouse spleen cells were used to evaluate the EBV gH / gL specific T cell immune response. EBV gp350FL LNP elicited strong T cell immune response, ELISpot analysis showed up to 500 IFN-y producing cells per million spleen cells, and demonstrated dose response (Figure I F). Immunization with gp470 LNP induced a similar T cell immune response as that of gp350 FL LNP (Figure IF).

[0428] Example 2: EBV gH / gL mRNA LNP vaccine candidates formulated with ARV-T1 induced potent antibody and T cell immune responses in BALB / c mice

[0429] EBV envelope proteins gH and gL play critical roles in EBV infection of its target cells through the fusion of tire virus envelope with tire cell membrane, which make them critical EBV vaccine candidates. 2 different EBV gH / gL mRNA LNP vaccine candidates were produced and characterized the immunogenicity of these vaccine candidates in BALB / c mice.

[0430] EBV gH and gL coding sequence were downloaded from Gene Bank, reference # NC_007605.1. DNA encoding EBV gL, gH, and gL-(G4S)j-gH with flanking 5’-UTR were Attorney Docket No. 11538-008W01 codon optimized and synthesized, and cloned into pUC57 vector that provided a 3’-UTR and poly A tail downstream. EBV mRNA encoding EBV gL, gH or gL-(G4S)3-gH was produced by in vitro transcription using an optimized T7 RNA polymerase- mediated transcription reaction with uridine-5* -triphosphate replaced by Nl-methylpseudouridine-5’-triphosphate, and a cap 1 structure was added enzymatically using vaccinia capping enzyme and 2’0-methyitransferase.

[0431] EBV lipid nanoparticle (LNP) formulations were prepared by mixing lipids in an organic phase (ethanol) with mRNA in an aqueous phase (50 mM citrate buffer, pH 4.0), maintaining a specific ratio (1:3, v / v) between the organic and aqueous phases. The total lipid concentration was 12.5 mM, with the molar percentage ratio for constituent lipids as: 50% ATV-T1 (ionizable lipids), 10%' DSPC, 38.5% cholesterol, and 1.5% DMG-PEG in ethanol. All formulations maintained a nitrogen-to -phosphate ratio (N / P) of 4, and the coformulation of gL and gH were conducted at 1:4 weight ratio to maintain 1: 1 molecular ration of gL to gH.

[0432] HEK-293T cells were transfected with LNP-mRNA encoding gH / gL formulated with ARV-T1, and the expression of gH / gL protein was evaluated by flow cytometry analysis using an EBV gH / gL antibody (eEnzyme, Cat# HHV4-gHL-308, Lot# I-129ES). Transfection of 293T cells with gH+gL or gL-fGrSh-gH mRNA using lipofectamine MessagerMax demonstrated >95% gH / gL positive cells as shown by flow cytometer analysis (Figure 2A and 2B). Similarly, LNPs encapsulating gH+gL (co-formulation) or gL-fGrSjj-gH (HLN5) mRNA showed >98% gH / gL positive cells, slightly higher than their corresponding mRNA counterparts (Figure 2 A and 2B).

[0433] For immunogenicity studies, BALB / c mice were immunized intramuscularly (quadriceps) with LNPs encapsulating gH / gL (coformulation) or gL-(G4S)3-gH formulated with ARV-T1 on day 1 (prime) and day 21 (boost), and PBS was injected as negative control. Seram was collected two weeks after prime and boost for binding antibody and EBV neutralizing antibody analysis. On day 35, mice were euthanized, and spleens were removed for IFN-y expression study with ELISpot analysis.

[0434] Immunization with EBV gH+gL LNP and gL-(G4>S)3-gH LNP both induced high titers of gH / gL-specific binding antibodies (103to 104) 21 days after 1stimmunization, and showed a trend dose response (Figure 2C). The titers of gH / gL binding antibodies increased at least 10- fold 35 days after 1stimmunization for both EBV gH+gL LNP and gL-(G4S)3-gH LNP as compared to their corresponding titers elicited 21 days after 1stimmunization. EBV gH+gL LNP 5(ug induced significantly higher gH / gL binding antibodies compared to 5(ug gL-(G4S)3-gH LNP 35 days after 1stimmunization (Figure 2C). Attorney Docket No. 11538-008W01

[0435] EB V-neutralizing antibody titers were determined in vitro using AKATA cells (EBV- negative human Burkitt lymphoma cell line) and EBV-GFP derived from CNE2 cells. Immunization with gH+gL LNP and gL~(G4S)3~gH LNP induced high titers of EBV neutralizing antibodies, in the range of 300 to 3000, and showed a dose response (Figure 2D). EBV gH+gL LNP induced slightly higher titers of EBV neutralizing antibodies compared to that of gL- (G4S)3-gH LNP, but were not statistically significant (Figure 2D).

[0436] Mouse spleen cells were used to evaluate the EBV gH / gL specific T cell immune response. Immunization with EBV gL-(G4S)3-gH LNP elicited strong T cell immune response, ELISpot analysis showed up to 1000 IFN-y producing cells per million spleen cells, and demonstrated clear dose response (Figure 2E). Immunization with gH+gL LNP Induced a similar T cell immune response as that of gL-(G4S)3-gH LNP (Figure 2E).

[0437] Example 3: EBV gB mRNA LNP vaccine candidates formulated with ARV-T1 induced high titers gB-specific antibodies and potent T cell immune response in BALB / c mice

[0438] EB V vaccine holds great promise for the prevention and treatment of infectious mononucleosis, multiple sclerosis, chronic periodontitis and multiple lymphoid and epithelial cancers. EBV envelope protein gB plays a critical role in EBV infection of its target cells through the fusion of the virus envelope with the cell membrane, and therefore is a key vaccine candidate for EBV vaccine. 3 different EBV gB mRNA LNP vaccine candidates were produced, and characterized the immunogenicity of these vaccine candidates in BALB / c mice.

[0439] EBV gB coding sequence was downloaded from Gene Bank, reference # NC_007605.L In addition to the wild type EBV gB (gBWT), two additional EBV gB constructs were designed, the post fusion EBV gB (gBPF) and the EBV gB with a (GljqSerh linker inserted at the furin cleavage site (gBLN). DNA encoding EBV gBWT, gBPF and gBLN with flanking 5’-UTR were codon optimized and synthesized, and cloned into pUC57 vector that provided a 3’-UTR and poly A tail downstream. EBV mRNA encoding EBV gBWT, gBPF or gBLN was produced by in vitro transcription using an optimized T7 RNA polymerase-mediated transcription reaction with uridine-5’ -triphosphate replaced by Nl-methylpseudouridine-5’ -triphosphate, and a cap 1 structure was added enzymatically using vaccinia capping enzyme and 2’O-methyltransferase.

[0440] EBV lipid nanoparticle (LNP) formulations were prepared by mixing lipids in an organic phase (ethanol) with mRNA in an aqueous phase (50 mM citrate buffer, pH 4.0), maintaining a specific ratio (1:3, v / v) between the organic and aqueous phases. The total lipid concentration was 12.5 mM, with the molar percentage ratio for constituent lipids as: 50% ATV-T1 (ionizable Attorney Docket No. 11538-008W01 lipids), 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG in ethanol. All formulations maintained a nitrogen-to-phosphate ratio (N / P) of 4.

[0441] HEK-293T cells were transfected with LNP-mRNA encoding gBWT, gBPF or gBLN formulated with ARV-T1, and the expression of gB protein was evaluated Western blot analysis using an EBV gB antibody (Cell Sciences, Newburyport MA). Transfection of 293T cells with EBV gBWT, gBLN or gBPF mRNA-LNP all showed strong EBV gB expression by Western blot analysis (Figure 3A).

[0442] For immunogenicity studies, BALB / c mice were immunized intramuscularly (quadriceps) with LNPs encapsulating gBWT, gBPF or gBLN formulated with ARV-T1 on day 1 (prime) and day 21 (boost), and PBS was injected as negative control. Serum was collected two weeks after prime and boost for binding antibody and EBV neutralizing antibody analysis. On day 35, mice were euthanized, and spleens were removed for IFN-y expression study with ELISpot analysis.

[0443] Immunization with EBV gBWT, gBLN or gBPF mRNA-LNP all induced high titers of gB-speciflc binding antibodies (104to 105) 21 days after ls!immunization, and demonstrated clear dose response (Figure 3B). The titers of gB binding antibodies increased about 10-fold 35 days after 1stimmunization for all the groups as compared to their corresponding titers elicited 21 days after ls!immunization (Figure 3B).

[0444] EBV-neutralizing antibody titers were determined in vitro using AKATA cells (EBV- negative human Burkitt lymphoma cell line) and EBV-GFP derived from CNE2 cells, immunization with high dose (5 pg) EBV gBWT, gBLN or gBPF mRNA-LNP induced moderate titers of EBV neutralizing antibodies, in the range of 150 to 200, and there was no difference when the 3 groups were compared (Figure 3C).

[0445] Mouse spleen cells were used to evaluate the EBV gH / gL specific T cell immune response. Immunization with 5 pg EBV gBWT, gBLN and gBPF mRNA-LNPs all induced strong T cell immune response in mice with high level IFN-v production, ELISpot analysis showed up to 500 IFN-v producing cells per million spleen cells (Figtire 3D).

[0446] Example 4: Profusion EBV gB mRNA LNP vaccine candidates formulated with ARV-T1 induced high titers EBV neutralizing antibodies and potent T cell immune response in BALB / c mice

[0447] EBV vaccine holds great promise for the prevention and treatment of infectious mononucleosis, multiple sclerosis, chronic periodontitis and multiple lymphoid and epithelial cancers. EBV envelope protein gB plays a critical role in EBV infection of its target cells Attorney Docket No. 11538-008W01 through the fusion of the virus envelope with the cell membrane, and therefore is a key vaccine candidate for EBV vaccine. The EBV gB in prefusion conformation is believed to induce higher neutralizing antibodies and is a perfect vaccine candidate. 3 prefusion EBV gB mRNA LNP vaccine candidates were produced, and characteri zed the immunogenicity of these vaccine candidates in BALB / c mice.

[0448] EBV gB coding sequence was downloaded from Gene Bank, reference # NC_ .007605.1. The prefusion EBV gB candidates were designed with AlphaFold 3 using prefusion CMV gB as the model and based on the structure of the EBV gB with a (GlyySerfj linker inserted at the furin cleavage site (gBLN). DNA encoding prefusion EBV gB (EBPRH, EBMPR and EBMPR-V) and gBLN (EBTMH) with flanking 5’-UTR were codon optimized and synthesized, and cloned into pUC57 vector that provided a 3’-UTR and poly A tail downstream. EBV mRNA encoding EBV prefusion gB or gBLN was produced by in vitro transcription using an optimi zed T7 RNA polymerase-mediated transcription reaction with uridine-5’ -triphosphate replaced by Nl- methylpseudouridine-5’ -triphosphate, and a cap 1 structure was added enzymatically using vaccinia capping enzyme and 2’0-methyltransferase.

[0449] EBV lipid nanoparticle (LNP) formulations were prepared by mixing lipids in an organic phase (ethanol) with mRNA in an aqueous phase (50 mM citrate buffer, pH 4.0), maintaining a specific ratio (1:3, v / v) between the organic and aqueous phases. The total lipid concentration was 12.5 mM, with the molar percentage ratio for constituent lipids as: 50% ATV-T1 (ionizable lipids), 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG in ethanol. All formulations maintained a nitrogen-to-phosphate ratio (N / P) of 4.

[0450] HEK-293T cells were transfected with LNP-mRNA encoding prefusion EBV gB or gBLN formulated with ARV-T1, and the expression of gB protein was evaluated Western blot analysis using an EBV gB antibody (Cell Sciences, Newburyport MA). Transfection of 293T cells with EBV EBPRH, EBMPR and EBMPR-V or EBTMH mRNA-LNP all showed strong EBV gB expression by Western blot analysis (Figure 4A).

[0451] For immunogenicity studies, BALB / c mice were immunized intramuscularly (quadriceps) with LNPs encapsulating prefusion EBV gB mRNA (EBPRH, EBMPR and EBMPR-V) or gBLN mRNA (EBTMH) formulated with ARV-T1 on day 1 (prime) and day 21 (boost), and PBS was injected as negative control. Serum was collected two weeks after prime and boost for binding antibody and EBV neutralizing antibody analysis. On day 35, mice were euthanized, and spleens were removed for IFN-y expression study with ELISpot analysis.

[0452] Immunization with EBV prefusion EBV gB or gBLN mRNA-LNP all induced high titers of gB-specific binding antibodies (106) 35 days after immunization, and there was no difference Attorney Docket No. 11538-008W01 when compare the EBV gB IgG induced by the prefusion EBV gB mRNA-LNP vaccine candidates (Figure 4B).

[0453] EBV-neutralizing antibody titers were determined in vitro using AKATA cells (EBV- negative human Burkitt lymphoma cell line) and EB V-GFP derived from CNE2 cells, immunization with low dose (1 pg) EBV gBLN mRNA-LNP induced base level EBV neutralizing antibodies, barely above lower limit of quantification (LLOQ). The prefusion EBV gB mRNA-LNP (EBPRH, EBMPR and EBMPR-V) vaccine candidates induced significantly higher EBV neutralizing antibodies, which was about 10-fold higher than the neutralizing antibodies induced by EBV gBLN mRNA-LNP (Figure 4C).

[0454] Mouse spleen cells were used to evaluate the EBV gH / gL specific T cell immune response. Immunization with 1 pg EBV prefusion EBV gB and gBLN mRNA-LNPs all induced strong T cell immune response in mice with high level IF'N-Y production, ELlSpot analysis showed up to 500 IFN-Y producing cells per million spleen cells (Figure 4D).

[0455] Additional prefusion EBV gB mRNA-LNPs have been produced (EBMSA2, EBMS9 and EBMSA10), and mouse immunization studies are being conducted (sequences provided).

[0456] Example 5: Co- Transfection of EBV gB mRNA LNP with gH / gL mRNA LNP as well as Co-Formulation of gB mRNA and gH / gL mRNA in a single LNP result in the expression of EB V gB / gH / gL complex

[0457] EBV vaccine holds great promise for the prevention and treatment of infectious mononucleosis, multiple sclerosis, chronic periodontitis and multiple lymphoid and epithelial cancers. Unlike the multiple latency programs, EBV lytic reactivation follows a single coordinated program, a progresses through distinct temporal phases: In the Immediate Early (IE) Phase, the first genes expressed upon reactivation include key transcription factors BZLF1 (Zta) often called the "lytic switch" protein and BRLF1 (Rta), both master regulators that initiate the entire lytic cascade. Early (E) Phase, DNA replication machinery and enzymes including viral DNA polymerase, primase, helicase, proteins involved in immune evasion such as BMRF1, BALF5, BALF2. The Late (L) Phase includes structural proteins for virion assembly including capsid proteins, tegument proteins, envelope glycoproteins, including the binding (gp350 / 220) and fusion proteins, as described below. Unlike the Latent Programs, there is essentially one lytic program that unfolds in a temporal cascade, unlike latency which has multiple distinct programs. The progression is tightly regulated, with each phase depending on the previous one. Attorney Docket No. 11538-008W01

[0458] EBV envelope protein gB and gH / gL play critical roles in EBV infection of its target cells through the fusion of the virus envelope with the cell membrane, and therefore are called ‘‘core fusion machinery”. The gB / gH / gL complex has been has been described (Nature Communications, 7:13557, DOI: 10.1038 / ncommsl3557) but a EBV gB / gH / gL complex as vaccine has not been described, given the complexity of the molecular entity and uncertainly that assembly could occur for presentation to the immune system . The core fusion machinery gB / gH / gL complex is considered as a superior vaccine candidate, as this complex can induce antibodies that recognize conformational epitopes with increased virus neutralizing activity. Cotransfection of cells with our EBV gB and gH / gL mRNA-LNP vaccine candidates or transfection of cells with the coformulation of EBV gB mRNA and gH / gL mRNA in a single LNP resulted in the expression of EBV gB / gH / gL protein complex. Immunogenicity studies of these vaccine candidates in BALB / c mice are being conducted.

[0459] EBV gB, gH and gL coding sequences were downloaded from Gene Bank, reference # NC„007605.1. DNA encoding prefusion EBV gB (EBPRH, EBPRM, EBMPR and EBMPR-V) and gBLN (EBTMH and EBNOP) with flanking 5’-UTR were codon optimized and synthesized, and cloned into pUC57 vector that provided a 3’-UTR and poly A tail downstream. EBV mRNA encoding EBV prefusion gB or gBLN was produced by in vitro transcription using an optimized T7 R NA polymer ase-mediated transcription reaction with uridine-5’ -triphosphate replaced by Nl-methylpseudouridine-5 ’-triphosphate, and a cap 1 structure was added enzymatically using vaccinia capping enzyme and 2’0-methyltransferase. EBV gL-(Gly4Ser)3-gH mRNA and gL- T2A-gH mRNA were produced similarly.

[0460] EBV lipid nanoparticle (LNP) formulations were prepared by mixing lipids ...

Claims

Attorney Docket No. 11538-008W01What is claimed is:

1. A composition comprising: a nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1, EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8, EBV gL-Linker-gH or a variant or fragment thereof including SEQ. ID. NO: 7, EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: II, EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ, ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof.

3. The composition of any one of claims 1-2, wherein the composition further comprises a nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EB V immediate early lytic protein or a variant or fragment thereof, wherein the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP-2A, EBV LMP-2B, EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1 , or a combination thereof.

4. The composition of claim 3, wherein the nucleic acid comprises a variant of EBV latent protein and / or EBV immediate early lytic protein including a deletion / mutation to one or more amino acid residues to eliminate oncogenic function.

5. The composition of any one of claims 3-4, wherein the nucleic acid comprises a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof.

6. The composition of claim 5, wherein the nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof comprises SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a combination thereof.Attorney Docket No. 11538-008W017. The composition of any one of claims 3-6, wherein the nucleic acid comprises a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof.

8. The composition of claim 7, wherein the nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof comprises SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a combination thereof.

9. The composition of any one of claims 3-8, wherein the nucleic acid comprises a nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof.

10. The composition of claim 9, wherein the nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof comprises SEQ. ID. NO: 16.

11. The composition of any one of claims 3-8, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof.

12. The composition of claim 11 , wherein the nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof comprises SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a combination thereof.

13. The composition of any one of claims 3-12, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof.

14. The composition of claim 13, wherein the nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof comprises SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 41, SEQ. ID. NO: 42, or a combination thereof.

15. The composition of any one of claims 3-14, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof.

16. The composition of claim 15, wherein the nucleic acid encoding for EBV EBN A-3A or a variant or fragment thereof comprises SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof.Attorney Docket No. 11538-008W0117. The composition of any one of claims 3-16, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof.

18. The composition of claim 17, wherein the nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof comprises SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a combination thereof.

19. The composition of any one of claims 3-18, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof.

20. The composition of claim 18, wherein the nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof comprises SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a combination thereof.

21. The composition of any one of claims 3-20, wherein the nucleic acid comprises a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

22. The composition of any one of claims 1-21, further comprising a nucleic acid encoding for a nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L (gL), EBV glycoprotein H (gH), EBV glycoprotein gp350, or a combination thereof.

23. The composition of claims 22, wherein the nucleic acid comprises a nucleic acid encoding for EBV gB or a variant or fragment thereof.

24. The composition of claim 23, wherein the nucleic acid encoding for EBV gB or a variant or fragment thereof comprises SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO:

32. SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof.

25. The composition of any one of claims 22-24, wherein the second nucleic acid comprises a nucleic acid encoding for EBV gfl or a variant or fragment thereof.Attorney Docket No. 11538-008W0126. The composition of claim 25, wherein the second nucleic acid encoding for EBV gH or a variant or fragment thereof comprises SEQ. ID. NO: 5.

27. The composition of any one of claims 22-26, wherein the second nucleic acid comprises a nucleic acid encoding for EBV gL or a variant or fragment thereof.

28. The composition of claim 27, wherein the second nucleic acid encoding for EBV gL or a variant or fragment thereof comprises SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, or a combination thereof.

29. The composition of any one of claims 22-28, wherein the second nucleic acid comprises a nucleic acid encoding for EBV gp350 or a variant or fragment thereof.

30. The composition of claim 29, wherein the second nucleic acid encoding for EBV gp350 or a variant or fragment thereof comprises SEQ. ID. NO: 9, SEQ. ID. NO: 10, or a combination thereof.

31. The composition of any one of claims 1-30, wherein the nucleic acid comprises plasmidDNA, minicircle DNA, microRNA, mRN A, self-amplifying RNA, circle RNA, DNA launched self-amplifying RNA, or viral vector.

32. A composition comprising: a nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof, wherein the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP-2A, EBV LMP-2B, EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1, or a combination thereof.

33. The composition of claim 32, wherein the nucleic acid comprises a variant of EBV latent protein and / or EBV immediate early lytic protein including a deletion / mutaiion to one or more amino acid residues to eliminate oncogenic function.Attorney Docket No. 11538-008W0134. The composition of any one of claims 32-33, wherein the nucleic acid comprises a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof.

35. The composition of claim 34, wherein the nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof comprises SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a combination thereof.

36. The composition of any one of claims 32.-35, wherein the nucleic acid comprises a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof.

37. The composition of claim 36, wherein the nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof comprises SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a combination thereof.

38. The composition of any one of claims 32-36, wherein the nucleic acid comprises a nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof.

39. The composition of claim 38, wherein the nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof comprises SEQ. ID. NO: 16.

40. The composition of any one of claims 32-39, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof.

41. The composition of claim 40, wherein the nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof comprises SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a combination thereof.

42. The composition of any one of claims 32-41 , wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof.

43. The composition of claim 42, wherein the nucleic acid encoding for EBV EBN A-2 or a variant or fragment thereof comprises SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 41, SEQ. ID. NO: 42, or a combination thereof.Attorney Docket No. 11538-008W0144. The composition of any one of claims 32-43, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof.

45. The composition of claim 44, wherein the nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof comprises SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof.

46. The composition of any one of claims 32.-45, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof.

47. The composition of claim 46, wherein the nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof comprises SEQ. ID. NO: 2.3, SEQ. ID. NO: 24, or a combination thereof.

48. The composition of any one of claims 32-47, wherein the nucleic acid comprises a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof.

49. The composition of claim 48, wherein the nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof comprises SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a combination thereof.

50. The composition of any one of claims 32-49, wherein the nucleic acid comprises a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

51. The composition of any one of claims 32-50, wherein the nucleic acid comprises plasmidDNA, minicircle DNA, microRNA, mRNA, self-amplifying RNA, circle RNA, DNA launched self-amplifying RNA, or viral vector.

52. A composition comprising: a first nucleic acid encoding for Epstein Barr virus (EBV) glycoprotein B (gB) glycoprotein BMRF2 or a variant or fragment thereof; andAttorney Docket No. 11538-008W01 a second nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L(gL), EBV glycoprotein II (gH), EBV glycoprotein gp350, or a combination thereof.

53. The composition of claim 52, wherein the first nucleic acid comprises SEQ, ID. NO: 11.

54. The composition of any one of claims 52-53, wherein the second nucleic acid comprises a nucleic acid encoding for EBV gB or a variant or fragment thereof.

55. The composition of claim 54, wherein the second nucleic acid encoding for EBV gB or a variant or fragment thereof comprises SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ. ID. NO: 4, SEQ, ID. NO: 29, SEQ. ID. NO: 30, SEQ, ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ, ID. NO: 37, SEQ. ID. NO: 38, SEQ, ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof.

56. The composition of any one of claims 52.-55, wherein the second nucleic acid comprises a nucleic acid encoding for EBV gH or a variant or fragment thereof.

57. The composition of claim 56, wherein the second nucleic acid encoding for EBV gH or a variant or fragment thereof comprises SEQ. ID. NO: 5.

58. The composition of any one of claims 52-57, wherein the second nucleic acid comprises a nucleic acid encoding for EBV gL or a variant or fragment thereof.

59. The composition of claim 58, wherein the second nucleic acid encoding for EBV gL or a variant or fragment thereof comprises SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, or a combination thereof.

60. The composition of any one of claims 52-59, wherein the second nucleic acid comprises a nucleic acid encoding for EBV gp350 or a variant or fragment thereof.

61. The composition of claim 60, wherein the second nucleic acid encoding for EBV gp350 or a variant or fragment thereof comprises SEQ. ID. NO: 9, SEQ. ID. NO: 10, or a combination thereof.Attorney Docket No. 11538-008W0162. The composition of any one of claims 52-61, wherein the first nucleic acid and the second nucleic acid comprise plasmid DNA, minicircle DNA, microRNA, mRNA, selfamplifying RNA, circle RN A, DNA launched self-amplifying RNA, or viral vector.

63. The composition of any one of claims 52-62, wherein the composition further comprises a third nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or anEBV immediate early lytic protein or a variant or fragment thereof, wherein the EBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1, EBV LMP-2A, EBV LMP-2B, EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1, or a combination thereof.

64. The composition of claim 63, wherein the third nucleic acid comprises a variant of EBV latent protein and / or EBV immediate early lytic protein including a deletion / mutation to one or more amino acid residues to eliminate oncogenic function.

65. The composition of any one of claims 63-64, wherein the third nucleic acid comprises a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof.

66. The composition of claim 65, wherein the third nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof comprises SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a combination thereof.

67. The composition of any one of claims 63-66, wherein the third nucleic acid comprises a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof.

68. The composition of claim 67, wherein the third nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof comprises SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a combination thereof.

69. The composition of any one of claims 63-68, wherein the third nucleic acid comprises a nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof.Attorney Docket No. 11538-008W0170. The composition of claim 69, wherein the third nucleic acid encoding for EB V LMP-2B or a variant or fragment thereof comprises SEQ. ID. NO: 16.71 . The composition of any one of claims 63-70, wherein the third nucleic acid comprises a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof.

72. The composition of claim 71, wherein the third nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof comprises SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a combination thereof.

73. The composition of any one of claims 63-72, wherein the third nucleic acid comprises a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof.

74. The composition of claim 73, wherein the third nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof comprises SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ. ID. NO: 41, SEQ. ID. NO: 42, or a combination thereof.

75. The composition of any one of claims 63-74, wherein the third nucleic acid comprises a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof.

76. The composition of claim 75, wherein the third nucleic acid encoding for EBV EBNA- 3A or a variant or fragment thereof comprises SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof.

77. The composition of any one of claims 63-76, wherein the third nucleic acid comprises a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof.

78. The composition of claim 77, wherein the third nucleic acid encoding for EBV EBNA- 3B or a variant or fragment thereof comprises SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a combination thereof.

79. The composition of any one of claims 63-78, wherein the third nucleic acid comprises a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof.Attorney Docket No. 11538-008W0180. The composition of claim 79, wherein the third nucleic acid encoding for EBV EBNA- 3C or a variant or fragment thereof comprises SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a combination thereof.81 . The composition of any one of claims 63-80, wherein the third nucleic acid comprises a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

82. The composition of any one of claims 63-81, wherein the third nucleic acid comprises plasmid DNA, minicircle DNA, microRNA, mRNA, self-amplifying RNA, circle RNA, DNA launched self-amplifying RNA, or viral vector.

83. A composition comprising: a first nucleic acid encoding for an Epstein Barr virus (EBV) envelope protein or a variant or fragment thereof; and a second nucleic acid encoding for an EBV latent protein or a variant or fragment thereof and / or an EBV immediate early lytic protein or a variant or fragment thereof.

84. The composition of claim 83, wherein the EBV envelope protein or a variant or fragment thereof is selected from the group consisting of EBV glycoprotein B (gB), EBV glycoprotein L (gL), EBV glycoprotein H (gH), EBV glycoprotein gp350, EBV glycoprotein BMRF2, or a combination thereof.

85. The composition of any one of claims 83-84, wherein the first nucleic acid comprises a nucleic acid encoding for EBV glycoprotein BMRF2.

86. The composition of claim 85, wherein the first nucleic acid encoding for EBV glycoprotein BMRF2 comprises SEQ. ID. NO: 11.

87. The composition of any one of claims 83-86, wherein the first nucleic acid comprises a nucleic acid encoding for EBV gB or a variant or fragment thereof.

88. The composition of claim 87, wherein the first nucleic acid encoding for EBV gB or a variant or fragment thereof comprises SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, SEQ.Attorney Docket No. 11538-008W01ID. NO: 4, SEQ, ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31 , SEQ. ID. NO: 32, SEQ. ID. NO:

33. SEQ. ID. NO: 34, SEQ. ID. NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof.

89. The composition of any one of claims 83-88, wherein the first nucleic acid comprises a nucleic acid encoding for EBV gH or a variant or fragment thereof.

90. The composition of claim 89, wherein the first nucleic acid encoding for EBV gH or a variant or fragment thereof comprises SEQ. ID. NO: 5.

91. The composition of any one of claims 83-90, wherein the first nucleic acid comprises a nucleic acid encoding for EBV gL or a variant or fragment thereof.

92. The composition of claim 91, wherein the first nucleic acid encoding for EBV gL or a variant or fragment thereof comprises SEQ. ID. NO: 6, SEQ. ID. NO: 7, SEQ. ID. NO: 8, or a combination thereof.

93. The composition of any one of claims §3-92, wherein the first nucleic acid comprises a nucleic acid encoding for EBV gp350 or a variant or fragment thereof.

94. The composition of claim 93, wherein the first nucleic acid encoding for EBV gp350 or a variant or fragment thereof comprises SEQ. ID. NO: 9, SEQ. ID. NO: 10, or a combination thereof.

95. The composition of any one of claims §3-94, wherein the first nucleic acid encodes for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of a nucleic acid encoding for an EBV envelope protein or a variant or fragment thereof selected from the group consisting of an EBV gBWT or a variant or fragment thereof including SEQ. ID. NO: 1, EBV gBPF or a variant or fragment thereof including SEQ. ID. NO: 2, EBV gL-T2A-gH or a variant or fragment thereof including SEQ. ID. NO: 8, EBV gL-Linker-gH or a variant or fragment thereof inchiding SEQ. ID. NO: 7 , EBV gp350 or a variant or fragment thereof including SEQ. ID. NO: 9, EBV BMRF2 or a variant or fragment thereof including SEQ. ID. NO: II, EBV prefusion gB or a variant or fragment thereof including SEQ. ID. NO: 29, SEQ. ID. NO: 30, SEQ. ID. NO: 31, SEQ. ID. NO: 32, SEQ. ID. NO: 33, SEQ. ID. NO: 34, SEQ. ID.Attorney Docket No. 11538-008W01NO: 35, SEQ. ID. NO: 36, SEQ. ID. NO: 37, SEQ. ID. NO: 38, SEQ. ID. NO: 39, SEQ. ID. NO: 40, or a combination thereof.

96. The composition of any one of claims 83-95, wherein the EJBV latent protein and / or EBV immediate early lytic protein is selected from the group consisting of EBV LMP-1 , EBV LMP- 2A, EBV LMP-2B, EBV EBNA-1, EBV EBNA-2, EBV EBNA-3A, EBV EBNA-3B, EBV EBNA-3C, EBV BZLF-1, or a combination thereof.

97. The composition of claim 96, wherein the second nucleic acid comprises a variant of EBV latent protein and / or EBV immediate early lytic protein including a deletion / mutation to one or more amino acid residues to eliminate oncogenic function.

98. The composition of any one of claims 83-97, wherein the second nucleic acid comprises a nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof.

99. The composition of claim 98, wherein the second nucleic acid encoding for EBV LMP-1 or a variant or fragment thereof comprises SEQ. ID. NO: 12, SEQ. ID. NO: 13, or a combination thereof.

100. The composition of any one of claims 83-99, wherein the second nucleic acid comprises a nucleic acid encoding for EBV LMP-2A or a variant or fragment thereof.

101. The composition of claim 100, wherein the second nucleic acid encoding for EBV LMP- 2A or a variant or fragment thereof comprises SEQ. ID. NO: 14, SEQ. ID. NO: 15, or a combination thereof.

102. The composition of any one of claims 83-101, wherein the second nucleic acid comprises a nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof.

103. The composition of claim 102, wherein the second nucleic acid encoding for EBV LMP-2B or a variant or fragment thereof comprises SEQ. ID. NO: 16.

104. The composition of any one of claims 83-103, wherein the second nucleic acid comprises a nucleic acid encoding for EBV EBNA-1 or a variant or fragment thereof.Attorney Docket No. 11538-008W01105. The composition of claim 104, wherein the second nucleic acid encoding for EBVEBNA-1 or a variant or fragment thereof comprises SEQ. ID. NO: 17, SEQ. ID. NO: 18, or a combination thereof.

106. The composition of any one of claims 83-105, wherein the second nucleic acid comprises a nucleic acid encoding for EBV EBNA-2 or a variant or fragment thereof.

107. The composition of claim 106, wherein the second nucleic acid encoding for EB VEBNA-2 or a variant or fragment thereof comprises SEQ. ID. NO: 19, SEQ. ID. NO: 20, SEQ.ID. NO: 41, SEQ. ID. NO: 42, or a combination thereof.

108. The composition of any one of claims 83-10'7, wherein the second nucleic acid comprises a nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof.

109. The composition of claim 108, wherein the second nucleic acid encoding for EBV EBNA-3A or a variant or fragment thereof comprises SEQ. ID. NO: 21, SEQ. ID. NO: 22, or a combination thereof.

110. The composition of any one of claims 83-109, wherein the second nucleic acid comprises a nucleic acid encoding for EBV EBNA-3B or a variant or fragment thereof.

111. The composition of claim 110, wherein the second nucleic acid encoding for EBVEBNA-3B or a variant or fragment thereof comprises SEQ. ID. NO: 23, SEQ. ID. NO: 24, or a combination thereof.

112. The composition of any one of claims 83-111, wherein the second nucleic acid comprises a nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof.

113. The composition of claim 112, wherein the second nucleic acid encoding for EBV EBNA-3C or a variant or fragment thereof comprises SEQ. ID. NO: 25, SEQ. ID. NO: 26, or a combination thereof.Attorney Docket No. 11538-008W01114. The composition of any one of claims 83-113, wherein the second nucleic acid comprises a nucleic acid encoding for EBV BZLF-1 or a variant or fragment thereof.

115. The composition of any one of claims 83-114, wherein the first nucleic acid and the second nucleic acid comprise plasmid DNA, minicircle DNA, microRNA, mRNA, selfamplifying RNA, circle RNA, DNA launched self-amplifying RNA, or viral vector.

116. The composition of any one of claims 1 -115, wherein the composition comprises a nanoparticle, a lipid nanoparticle dispersion, a liposomal formulation, a lipid emulsion, vaccine, vector, or any combination thereof.

117. The composition of any one of claims 1-116, wherein the composition is a lipid nanoparticle, and wherein the lipid nanoparticle comprises 20% to 80% of ionizable lipid, cationic lipid, or any combination thereof; 0 % to 5 % pegylated lipids; 0 % to 40 % helper lipids: and 0 % to 80 % sterol.

118. The composition of any one of claims 1 -117, wherein the composition comprises a pharmaceutical composition suitable for administration to a human subject.

119. A nucleic acid comprises SEQ. ID. NO: 4.

120. A cell comprising the nucleic acid of claim 119 or the composition of any one of claims1-118.

121. The cell of claim 120, wherein the cell comprises a dendritic cell, B cell, or a macrophage.

122. A method of treating or preventing EBV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-118 or the nucleic acid of 119.

123. A method of preventing a disease or disorder caused by EBV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-118 or the nucleic acid of 119.Attorney Docket No. 11538-008W01124. A method of treating a disease or disorder caused by EBV infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1 -118 or the nucleic acid of 119.

125. The method of any one of claims 123-124, wherein the disease or disorder caused by EBV infection is selected from the group consisting of chronic periodontitis, EBV-associated cancer, multiple sclerosis, post-transplantation lymphoproliferative disease, chronic immune dysregulation, rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, and combinations thereof.

126. The method of claim 125, wherein the EBV-associated cancer is selected from lymphoma, nasopharyngeal carcinoma, and gastric carcinoma.

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